Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating Same

ABSTRACT

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses a single light chain variable domain derived from a single rearranged light chain variable region gene in the germline of the non-human animal, wherein the single rearranged light chain variable region gene comprises a substitution of at least one non-histidine encoding codon with a histidine encoding codon. Methods of making non-human animals that express antibodies comprising a histidine-containing universal light chain are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/339,376, filed Jul. 23, 2014, which is a divisional applicationof U.S. application Ser. No. 13/832,247, filed Mar. 15, 2013, now U.S.Pat. No. 9,334,334, issued May 10, 2016, which claims benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 61/611,950, filedMar. 16, 2012, and U.S. Provisional Application No. 61/736,930, filedDec. 13, 2012, all incorporated by reference herein in their entireties.

SEQUENCE LISTING

An official copy of the sequence listing is submitted concurrently withthe specification electronically via EFS-Web as an ASCII formattedsequence listing with a file name of2016-07-14-126000N1-US-SEQ-LIST_ST25.txt, a creation date of Jul. 14,2016, and a size of about 30.9 kilobytes. The sequence listing containedin this ASCII formatted document is part of the specification and isherein incorporated by reference in its entirety.

FIELD OF INVENTION

A genetically modified non-human animal (e.g., rodent, e.g., mouse orrat) is provided that expresses antibodies capable of binding to anantigen in a pH dependent manner. A method for making modifications toimmunoglobulin light chain variable region sequence of a non-humananimal is provided, wherein the modifications include the mutagenesis ofresidues within the light chain variable region gene, e.g., nucleotidesthat encode one or more amino acids within a complementary determiningregion (CDR), to facilitate in vivo expression of antibodies comprisinglight chain domains that exhibit pH dependent binding to antigens.Methods for making antibodies with pH-dependent antigen binding are alsoprovided.

BACKGROUND OF THE INVENTION

Antibodies typically comprise a homodimeric heavy chain component,wherein each heavy chain monomer is associated with an identical lightchain. Antibodies having a heterodimeric heavy chain component (e.g.,bispecific antibodies) are desirable as therapeutic antibodies. Butmaking bispecific antibodies having a suitable light chain componentthat can satisfactorily associate with each of the heavy chains of abispecific antibody has proved problematic.

In one approach, a light chain might be selected by surveying usagestatistics for all light chain variable domains, identifying the mostfrequently employed light chain in human antibodies, and pairing thatlight chain in vitro with the two heavy chains of differing specificity.

In another approach, a light chain might be selected by observing lightchain sequences in a phage display library (e.g., a phage displaylibrary comprising human light chain variable region sequences, e.g., ahuman scFv library) and selecting the most commonly used light chainvariable region from the library. The light chain can then be tested onthe two different heavy chains of interest.

In another approach, a light chain might be selected by assaying a phagedisplay library of light chain variable sequences using the heavy chainvariable sequences of both heavy chains of interest as probes. A lightchain that associates with both heavy chain variable sequences might beselected as a light chain for the heavy chains.

In another approach, a candidate light chain might be aligned with theheavy chains' cognate light chains, and modifications are made in thelight chain to more closely match sequence characteristics common to thecognate light chains of both heavy chains. If the chances ofimmunogenicity need to be minimized, the modifications preferably resultin sequences that are present in known human light chain sequences, suchthat proteolytic processing is unlikely to generate a T cell epitopebased on parameters and methods known in the art for assessing thelikelihood of immunogenicity (i.e., in silico as well as wet assays).

All of the above approaches rely on in vitro methods that subsume anumber of a priori restraints, e.g., sequence identity, ability toassociate with specific pre-selected heavy chains, etc. There is a needin the art for compositions and methods that do not rely on manipulatingin vitro conditions, but that instead employ more biologically sensibleapproaches to making human epitope-binding proteins that include acommon light chain.

In addition, therapeutic antibodies, e.g., bispecific therapeuticantibodies, have some limitations in that they often require high dosesto achieve desired efficacy. This is partly due to the fact thatantibody-antigen complexes are internalized into the endosome, and aretargeted for lysosomal degradation in a process called target-mediatedclearance. Thus, there is a need in the art for methods and compositionsthat lead to more efficient antibody recycling, e.g., bispecificantibody recycling, and prevent degradation of the antibody by promotingdissociation of antibody-antigen complexes in the endosomal compartmentwithout compromising the specificity and affinity of the antibody towardthe antigen.

SUMMARY OF THE INVENTION

In one aspect, a biological system is provided for generating anantibody or an antibody variable domain that binds a target antigen at aneutral pH but exhibits reduced binding of the same antigen at an acidicpH (e.g., pH 5.0-6.0). The biological system comprises a non-humananimal, e.g., a rodent (e.g., a mouse or rat) that has a rearrangedlight chain sequence (e.g., a rearranged V-J) that comprises one or morehistidine modifications. In various aspects, the one or more histidinemodifications are in the light chain CDR3 codon. In various aspects, thenon-human animal comprises a human or humanized heavy chainimmunoglobulin locus. In various aspects, the non-human animal comprisesa replacement of endogenous non-human heavy chain variable gene segmentswith one or more human heavy chain V_(H), D_(H), and J_(H) segments,wherein the human segments are operably linked to a non-humanimmunoglobulin constant region. In various aspects, non-human animalswith universal light chains comprising light chain variable domains withsubstitutions of non-histidine residues for histidine residues areprovided. In various aspects these histidine-modified universal lightchain non-human animals (e.g., rodents, e.g., mice) are referred to ashistidine-universal light chain mice, histidine-ULC mice, or HULC mice.

Thus, in one aspect, provided herein is a genetically modified non-humananimal that comprises in its germline an immunoglobulin light chainlocus that comprises a single rearranged human immunoglobulin lightchain variable region gene sequence comprising human V_(L) and J_(L)segment sequences, wherein the single rearranged human immunoglobulinlight chain variable region sequence comprises a substitution of atleast one non-histidine codon with a histidine codon. In one embodiment,the single rearranged human immunoglobulin variable region sequence isoperably linked to an immunoglobulin light chain constant region genesequence. In one embodiment, the immunoglobulin light chain constantregion gene sequence is a non-human immunoglobulin light chain constantregion gene sequence. In one embodiment, the non-human immunoglobulinlight chain constant region gene sequence is an endogenousimmunoglobulin light chain constant region gene sequence. In oneembodiment, the non-human animal lacks a functional unrearrangedimmunoglobulin light chain variable region. In one embodiment, theimmunoglobulin light chain locus is at an endogenous non-humanimmunoglobulin light chain locus.

In one embodiment, the animal further comprises in its germline animmunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region gene sequence comprisinghuman V_(H), D_(H), and J_(H) segments operably linked to animmunoglobulin heavy chain constant region gene sequence. In oneembodiment, the immunoglobulin heavy chain constant region gene sequenceis a non-human heavy chain constant region gene sequence. In oneembodiment, the non-human heavy chain constant region gene sequence isan endogenous immunoglobulin heavy chain constant region gene sequence.In one embodiment, the immunoglobulin heavy chain locus is at anendogenous immunoglobulin heavy chain locus.

In one embodiment, the substitution of at least one non-histidine codonwith a histidine codon is in the nucleotide sequence encoding acomplementary determining region (CDR). In one embodiment, thesubstitution of at least one non-histidine codon with a histidine codonis in the nucleotide sequence encoding a CDR3. In one embodiment, thesubstitution is of one, two, three, four, or more CDR3 codons. In oneaspect, the single rearranged human immunoglobulin light chain variableregion sequence comprised at the immunoglobulin light chain locus isderived from a human Vκ1-39 or Vκ3-20 gene segment. In one embodiment,the single rearranged human immunoglobulin light chain variable regionis derived from a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 gene sequence. Inone embodiment, the single rearranged human immunoglobulin light chainvariable region is derived from a rearranged Vκ1-39/Jκ5 gene sequence,and the Vκ1-39/Jκ5 gene sequence comprises a replacement of at least onenon-histidine codon with a histidine codon designed to express ahistidine at a position selected from 105, 106, 108, 111, and acombination thereof. In another embodiment, the single rearranged humanimmunoglobulin light chain variable region is derived from a rearrangedVκ3-20/Jκ1 gene sequence, and the Vκ3-20/Jκ1 gene sequence comprises areplacement of at least one non-histidine codon with a histidine codondesigned to express a histidine at a position selected from 105, 106,107, 109, and a combination thereof.

In one aspect, the non-human animal described herein comprises apopulation of B cells in response to an antigen of interest that isenriched for antibodies that exhibit a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.In one embodiment, the decrease in t_(1/2) at an acidic pH as comparedto a neutral pH is about 30 fold or more.

In one embodiment, the animal expresses an antibody comprising a humanimmunoglobulin light chain variable domain with a substitution of atleast one non-histidine residue with a histidine residue at an aminoacid position encoded by the at least one codon substituted in theimmunoglobulin light chain variable region gene sequence. In oneembodiment, the animal expresses an antibody that retains a substitutionof at least one non-histidine residue with a histidine residue in anexpressed human immunoglobulin light chain variable domain, despitesomatic hypermutations.

In one embodiment, the non-human animal is a mammal. In one embodiment,the mammal is a rodent, e.g., a rat or a mouse. In one embodiment, thenon-human animal is a mouse. Thus, in one aspect, also provided hereinis a genetically modified mouse comprising in its germline animmunoglobulin light chain locus that comprises a single rearrangedhuman immunoglobulin light chain variable region gene sequencecomprising human V_(L) and J_(L) segment sequences, wherein the singlerearranged human immunoglobulin light chain variable region sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon. In one embodiment, the mouse lacks a functionalunrearranged immunoglobulin light chain variable region.

In one embodiment, the single rearranged immunoglobulin light chainvariable region gene sequence in the germline of the mouse is operablylinked to an immunoglobulin light chain constant region gene sequence.In one embodiment, the immunoglobulin light chain constant region genesequence is selected from a rat or a mouse immunoglobulin light chainconstant region gene sequence. In one embodiment, the immunoglobulinlight chain constant region gene sequence is a mouse sequence. In oneembodiment, the immunoglobulin light chain locus is at an endogenousmouse immunoglobulin light chain locus.

In a further embodiment, the mouse also comprises in its germline animmunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region sequence comprising humanV_(H), D_(H), and J_(H) segments operably linked to an immunoglobulinheavy chain constant region gene sequence. In one aspect, theimmunoglobulin heavy chain constant region gene sequence is a rat or amouse heavy chain constant region gene sequence. In one embodiment, theimmunoglobulin heavy chain constant region gene sequence is a mousesequence. In one embodiment, the immunoglobulin heavy chain locus is atan endogenous mouse immunoglobulin heavy chain locus.

In one aspect, the mouse comprises a substitution of at least onenon-histidine codon with a histidine codon wherein the substitution isin the nucleotide sequence encoding a CDR. In one embodiment, thesubstitution is in a CDR3 codon, e.g., in one, two, three, four, or moreCDR3 codons. In one embodiment, the immunoglobulin light chain locus ofthe mouse comprises the single rearranged human immunoglobulin lightchain variable region sequence derived from a human Vκ1-39 or Vκ3-20gene segment, e.g., the single rearranged immunoglobulin light chainvariable region sequence is derived from a rearranged Vκ1-39/Jκ5 orVκ3-20/Jκ1 gene sequence. In one embodiment, the single rearrangedimmunoglobulin light chain variable region sequence is derived from arearranged Vκ1-39/Jκ5 gene sequence and the Vκ1-39/Jκ5 sequencecomprises a replacement of at least one non-histidine codon with ahistidine codon designed to express a histidine at a position selectedfrom 105, 106, 108, 111, and a combination thereof. In one embodiment,such replacement is designed to replace histidines at positions 105,106, 108, and 111. In another embodiment, such replacement is designedto replace histidines at positions 106, 108, and 111.

In another embodiment, the single rearranged immunoglobulin light chainvariable region sequence is derived from a rearranged Vκ3-20/Jκ1 genesequence and the Vκ3-20/Jκ1 sequence comprises a replacement of at leastone non-histidine codon with a histidine codon designed to express ahistidine at a position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, such replacement is designed toreplace histidines at positions 105, 106, 107, and 109. In anotherembodiment, such replacement is designed to replace histidines atpositions 105, 106, and 109.

In one embodiment, the mouse described herein comprises a population ofB cells in response to an antigen of interest that is enriched forantibodies that exhibit a decrease in dissociative half-life (t_(1/2))at an acidic pH as compared to neutral pH of at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 25-fold, or at least about 30-fold. In one embodiment, thedecrease in t_(1/2) at an acidic pH as compared to a neutral pH is about30 fold or more.

In one embodiment, the mouse described herein expresses a population ofantigen-specific antibodies in response to an antigen of interestwherein all antibodies comprise (a) immunoglobulin light chain variabledomains derived from the same single rearranged human light chainvariable region gene sequence which comprises a substitution of at leastone non-histidine codon with a histidine codon, and (b) immunoglobulinheavy chains comprising heavy chain variable domains derived from arepertoire of human heavy chain V, D, and J segments.

Also provided herein is a non-human locus, e.g., mouse locus, comprisinga single rearranged human immunoglobulin light chain variable regiongene sequence comprising human V_(L) and J_(L) segment sequences,wherein the single rearranged human immunoglobulin light chain variableregion gene sequence comprises a substitution of at least onenon-histidine codon with a histidine codon. In one embodiment, the locusis comprised in the germline of a non-human animal. In one embodiment,the locus comprises the single rearranged human immunoglobulin lightchain variable region gene sequence derived from a human Vκ1-39 orVκ3-20 gene segment, e.g., derived from a rearranged Vκ1-39/Jκ5 orVκ3-20/Jκ1 gene sequence. In one embodiment, wherein the singlerearranged human immunoglobulin light chain variable region genesequence present in the locus is derived from the rearranged Vκ1-39/Jκ5sequence, the substitution of at least one non-histidine codon with ahistidine codon is designed to express a histidine at a positionselected from 105, 106, 108, 111, and a combination thereof. In anotherembodiment, wherein the single rearranged human immunoglobulin lightchain variable region gene sequence present in the locus is derived fromthe rearranged Vκ3-20/Jκ1 sequence, the substitution of at least onenon-histidine codon with a histidine codon is designed to express ahistidine at a position selected from 105, 106, 107, 109, and acombination thereof. In various embodiments, the non-human locidescribed herein may be generated using methods described below formaking a genetically modified non-human animal.

In yet another aspect, provided herein is a method for making anon-human animal that comprises a genetically modified immunoglobulinlight chain locus in its germline, wherein the method comprisesmodifying a genome of a non-human animal to delete or rendernonfunctional endogenous immunoglobulin light chain V and J segments inan immunoglobulin light chain locus, and placing in the genome a singlerearranged human light chain variable region gene sequence comprising asubstitution of at least one non-histidine codon with a histidine codon.In one embodiment, such method results in a genetically modifiednon-human animal that comprises a population of B cells enriched forantibodies exhibiting pH-dependent binding to the antigen of interest.In one embodiment, the single rearranged human immunoglobulin lightchain variable region sequence placed in the genome is derived from ahuman Vκ1-39 or Vκ3-20, e.g., a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 genesequence. Thus, in the embodiment wherein the single rearranged humanimmunoglobulin light chain variable region sequence is derived from arearranged Vκ1-39/Jκ5, the substitution of at least one non-histidinecodon with a histidine codon is designed to express a histidine at aposition selected from 105, 106, 108, 111, and a combination thereof. Inan embodiment wherein the single rearranged human immunoglobulin lightchain variable region sequence is derived from a rearranged Vκ3-20/Jκ1,the substitution of at least one non-histidine codon with a histidinecodon is designed to express a histidine at a position selected from105, 106, 107, 109, and a combination thereof.

In another aspect, provided herein is a method of generating an antibodythat exhibits pH-dependent binding to an antigen of interest comprising(a) generating a mouse described herein (e.g., a mouse that comprises inits germline an immunoglobulin light chain locus that comprises a singlerearranged human immunoglobulin light chain variable region sequencecomprising human V_(L) and J_(L) segment sequences and a substitution ofat least one non-histidine codon with a histidine codon in itsrearranged light chain variable region sequence), (b) immunizing themouse with an antigen of interest, and (c) selecting an antibody thatbinds to the antigen of interest with a desired affinity at a neutral pHwhile displaying reduced binding to the antigen at an acidic pH. In oneembodiment, the method results in a generation of an antibody thatexhibits t_(1/2) at acidic pH and 37° C. of about 2 minutes or less. Inone embodiment, the method results in a generation of an antibody thatdisplays a decrease in dissociative half-life (t_(1/2)) at an acidic pHas compared to neutral pH of at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about25-fold, or at least about 30-fold.

In other aspects, provided herein are additional methods of generatingan antibody that exhibits pH-dependent binding to an antigen ofinterest. One such method comprises (a) selecting a first antibody thatbinds to an antigen of interest with a desired affinity, (b) modifyingan immunoglobulin light chain nucleotide sequence of the first antibodyto comprise a substitution of at least one non-histidine codon with ahistidine codon, (c) expressing an immunoglobulin heavy chain of thefirst antibody and the modified immunoglobulin light chain in a cell,and (d) selecting a second antibody expressed in the cell that retains adesired affinity for the antigen of interest at neutral pH and displaysreduced binding to the antigen of interest at an acidic pH. In oneembodiment, the immunoglobulin light chain nucleotide sequence of thefirst antibody comprises a single rearranged human immunoglobulin lightchain variable region sequence. In one embodiment, the first antibody isgenerated in a non-human animal, e.g., a mouse, comprising animmunoglobulin light chain sequence derived from a single rearrangedhuman immunoglobulin light chain variable region sequence, and themodification of the immunoglobulin light chain is made in the singlerearranged human immunoglobulin variable region sequence. In oneembodiment, the first antibody is generated in a non-human animal, e.g.,a mouse, further comprising an immunoglobulin heavy chain sequencederived from a repertoire of human V_(H), D_(H), and J_(H) segments. Inone embodiment, the single rearranged human immunoglobulin light chainvariable region sequence is selected from Vκ1-39/Jκ5 and Vκ3-20/Jκ1 genesequence. In an embodiment, wherein the single rearranged humanimmunoglobulin light chain variable region sequence is Vκ1-39/Jκ5, themodification in the immunoglobulin light chain nucleotide sequence ofthe first antibody is made in the CDR3 codon at a position selected from105, 106, 108, 111, and a combination thereof. In an embodiment whereinthe single rearranged human immunoglobulin light chain variable regionsequence is Vκ3-20/Jκ1, the modification in the immunoglobulin lightchain nucleotide sequence of the first antibody is made in the CDR3codon at a position selected from 105, 106, 107, 109, and a combinationthereof.

In one embodiment, the method of generating an antibody that exhibitspH-dependent binding to an antigen of interest described herein resultsin an antibody that displays a decrease in dissociative half-life(t_(1/2)) at an acidic pH as compared to neutral pH of at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold. In oneembodiment, the method of generating the antibody results in an antibodythat exhibits a t_(1/2) at acidic pH and 37° C. of about 2 minutes orless.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an amino acid alignment of human Vκ1-39-derived lightchains from various antigen-specific antibodies (A-K antibodies,corresponding to SEQ ID NOs: 82-92, respectively). Histidine (H)residues located within each light chain sequence are in bold. Variouslight chain regions (Framework and CDR) are indicated above thealignment.

FIG. 2 illustrates the combinations and locations of histidine residuesengineered in the CDR3 region of human Vκ1-39-derived light chains bymutagenesis. Corresponding nucleic acid sequences are included.Histidine residues introduced through mutagenesis and correspondingnucleic acid residues are shown in bold. Amino acid positions (105, 106,etc.) are based on a unique numbering described in Lefranc et al. (2003)Dev. Comp. Immunol. 27:55-77, and can also be viewed on the website ofthe International Immunogenetics Information System (IMGT).

FIG. 3 illustrates the level of antibody expression in ng/mL detected inthe supernatants of CHO cells transfected with nucleic acids encodingfive (1-5) different heavy chains and Vκ1-39-derived light chains havinghistidine residues engineered at indicated locations (see Y axis) in theCDR3.

FIG. 4 is a western blot showing expression of selected antigen-specifichuman antibodies containing histidine engineered light chains in CHOcell supernatants.

FIGS. 5A-5E show the binding kinetics for selected heavy chains (1-5)from antigen-specific antibodies paired with various histidineengineered light chains at a neutral (7.4) and acidic (5.75) pH. Variouskinetic parameters including k_(a), k_(d), K_(D), and t_(1/2) are shown.NB=no binding.

FIG. 6 shows kinetic parameters (K_(D) and t_(1/2)) for antibodiescomprising parental universal light chain or histidine-modifieduniversal light chain paired with indicated heavy chains (2, 3, and 6).Histidine substitutions lead to strong pH dependence in severalantibodies. Histidine substitutions were made in CDR3 to convert thesequence ₁₀₅QQSYSTP₁₁₁ (SEQ ID NO:3) to ₁₀₅HHSYSTH₁₁₁ (SEQ ID NO:5).Note that NB=no binding detected (KD>10 micromolar).

FIG. 7 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer histidine residuesinto CDR3 of a rearranged human Vκ1-39/Jκ5 light chain sequence. SEQ IDNOs for these primers used in the Sequence Listing are included in theTable below. F=forward primer, R=reverse primer.

FIGS. 8A-8B show a general strategy for construction of targetingvectors for engineering of histidine residues into a rearranged humanlight chain variable region sequence derived from Vκ1-39/Jκ5 variableregion for making a genetically modified mouse that expresses antibodiescontaining the modified human light chain. FIGS. 8C-8D show introductionof the targeting vector for ULC-H105/106/108/111 substitutions into EScells and generation of heterozygous mice from the same; while FIGS.8E-8F show introduction of the targeting vector for ULC-H106/108/111substitutions into ES cells and generation of heterozygous mice from thesame. The diagrams are not presented to scale. Unless indicatedotherwise, filled shapes and solid lines represent mouse sequence, emptyshapes and double lines represent human sequence.

FIG. 9 shows antiserum titers against immunogen from mice heterozygousfor histidine universal light chain (HULC) (with 4 Hissubstitutions—HULC 1927 mice; with 3 His substitutions—HULC 1930 mice)and wild type animals in a second bleed.

FIG. 10 is a comparison of the number of total antigen positive clonesand the number of antigen positive clones displaying pH sensitiveantigen binding obtained from hybridoma fusions from heterozygous HULC(1927 vs 1930) and WT mice. Figure includes data for two mice for eachmouse type (“mouse 1” and “mouse 2”).

FIGS. 11A-11C show sensorgrams from surface plasmon resonance bindingexperiments in which monoclonal antibodies (AA, BB, CC, DD, HH, GG, NN,and 00) from either heterozygous HULC or WT mice were allowed toassociate with the immunogen at neutral pH (pH 7.4) followed by a shiftto a buffer with pH of either 7.4 or 6.0 for the dissociation phase. Theindividual lines in each graph represent the binding responses atdifferent concentrations of the respective antibodies. All experimentswere carried out at 25° C. Dissociative half-life values (t½) are notedabove the respective sensorgrams, and fold change in t½ is included tothe right of each sensorgram. Antibodies AA, BB, CC, DD, HH, and GG werefrom heterozygous HULC 1927 mice using His-substituted light chain, NNis from heterozygous HULC 1927 mouse using WT light chain, and OO isfrom a WT mouse (See Table 4 for clarification).

FIG. 12 shows positions of histidine residues engineered in the CDR3region of human Vκ3-20-derived light chains by mutagenesis. Histidineresidues introduced through mutagenesis and corresponding nucleic acidresidues are shown in bold. Amino acid positions (105, 106, etc.) arebased on a unique numbering described in Lefranc et al. (2003) Dev.Comp. Immunol. 27:55-77, and can also be viewed on the website of theInternational Immunogenetics Information System (IMGT).

FIG. 13 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer histidine residuesinto CDR3 of a rearranged human Vκ3-20/Jκ1 light chain sequence. SEQ IDNOs for these primers used in the Sequence Listing are included in theTable below. F=forward primer, R=reverse primer.

FIGS. 14A-14B show a general strategy for construction of targetingvectors for the engineering of histidine residues into a rearrangedhuman light chain variable region sequence derived from Vκ3-20/Jκ1 lightchain variable region for making a genetically modified mouse thatexpresses antibodies containing the modified human light chain. FIG. 14Cshows introduction of the targeting vector forULC-Q105H/Q106H/Y107H/S109H substitutions into ES cells and generationof heterozygous mice from the same; while FIG. 14D shows introduction ofthe targeting vector for ULC-Q105H/Q106H/S109H substitutions into EScells and generation of heterozygous mice from the same. The diagramsare not presented to scale. Unless indicated otherwise, filled shapesand solid lines represent mouse sequence, empty shapes and double linesrepresent human sequence.

DETAILED DESCRIPTION OF INVENTION Definitions

The present invention provides genetically modified non-human animals(e.g., mice, rats, rabbits, hamsters, etc.) that comprise in theirgenome, e.g., in their germline, nucleotide sequence(s) encoding humanantibody molecules that exhibit pH-dependent antigen binding, e.g., anucleotide sequence of immunoglobulin light chain comprising rearrangedhuman immunoglobulin light chain variable region sequence encodingantibodies that exhibits pH-dependent antigen binding; embryos, cells,and tissues comprising the same; methods of making the same; as well asmethods of using the same. Unless defined otherwise, all terms andphrases used herein include the meanings that the terms and phrases haveattained in the art, unless the contrary is clearly indicated or clearlyapparent from the context in which the term or phrase is used.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable domain and a heavy chain constantregion (C_(H)). The heavy chain constant region comprises three domains,C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chainvariable domain and a light chain constant region (CO. The heavy chainand light chain variable domains can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each heavy and light chain variable domain comprises threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chainCDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs maybe abbreviated as LCDR1, LCDR2 and LCDR3). The term “high affinity”antibody refers to an antibody that has a K_(D) with respect to itstarget epitope about of 10⁻⁹ M or lower (e.g., about 1×10⁻⁹ M, 1×10⁻¹⁰M, 1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment, K_(D) is measuredby surface plasmon resonance, e.g., BIACORE™; in another embodiment,K_(D) is measured by ELISA.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two nonidentical heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., different epitopes on two different immunogens) or onthe same molecule (e.g., different epitopes on the same immunogen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst heavy chain for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Exemplary bispecific antibodies include those with a firstheavy chain specific for a tumor antigen and a second heavy chainspecific for a cytotoxic marker, e.g., an Fc receptor (e.g., FcγRI,FcγRII, FcγRIII, etc.) or a T cell marker (e.g., CD3, CD28, etc.).Further, the second heavy chain variable domain can be substituted witha heavy chain variable domain having a different desired specificity.For example, a bispecific antibody with a first heavy chain specific fora tumor antigen and a second heavy chain specific for a toxin can bepaired so as to deliver a toxin (e.g., saporin, vinca alkaloid, etc.) toa tumor cell. Other exemplary bispecific antibodies include those with afirst heavy chain specific for an activating receptor (e.g., B cellreceptor, FcγRI, FcγRIIA, FcγRIIIA, FcαRI, T cell receptor, etc.) and asecond heavy chain specific for an inhibitory receptor (e.g., FcγRIIB,CD5, CD22, CD72, CD300a, etc.). Such bispecific antibodies can beconstructed for therapeutic conditions associated with cell activation(e.g. allergy and asthma). Bispecific antibodies can be made, forexample, by combining heavy chains that recognize different epitopes ofthe same immunogen. For example, nucleic acid sequences encoding heavychain variable sequences that recognize different epitopes of the sameimmunogen can be fused to nucleic acid sequences encoding the same ordifferent heavy chain constant regions, and such sequences can beexpressed in a cell that expresses an immunoglobulin light chain. Atypical bispecific antibody has two heavy chains each having three heavychain CDRs, followed by (N-terminal to C-terminal) a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain, and an immunoglobulin lightchain that either does not confer epitope-binding specificity but thatcan associate with each heavy chain, or that can associate with eachheavy chain and that can bind one or more of the epitopes bound by theheavy chain epitope-binding regions, or that can associate with eachheavy chain and enable binding of one or both of the heavy chains to oneor both epitopes.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, W138, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. A CDR can be somatically mutated(e.g., vary from a sequence encoded in an animal's germline), humanized,and/or modified with amino acid substitutions, additions, or deletions.In some circumstances (e.g., for a CDR3), CDRs can be encoded by two ormore sequences (e.g., germline sequences) that are not contiguous (e.g.,in an unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as the result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of avariable region to specifically bind a target epitope with a desiredaffinity. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be substitution of any native residue in a protein withalanine, as used in, for example, alanine scanning mutagenesis. In someembodiments, a conservative substitution is made that has a positivevalue in the PAM250 log-likelihood matrix disclosed in Gonnet et al.(1992) Exhaustive Matching of the Entire Protein Sequence Database,Science 256:1443-45, hereby incorporated by reference. In someembodiments, the substitution is a moderately conservative substitutionwherein the substitution has a nonnegative value in the PAM250log-likelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chainor heavy chain differ by one or more conservative amino acidsubstitutions. In some embodiments, residue positions in animmunoglobulin light chain or functional fragment thereof (e.g., afragment that allows expression and secretion from, e.g., a B cell) arenot identical to a light chain whose amino acid sequence is listedherein, but differs by one or more conservative amino acidsubstitutions.

The phrase “epitope-binding protein” includes a protein having at leastone CDR and that is capable of selectively recognizing an epitope, e.g.,is capable of binding an epitope with a K_(D) that is at about onemicromolar or lower (e.g., a K_(D) that is about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁹ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).Therapeutic epitope-binding proteins (e.g., therapeutic antibodies)frequently require a K_(D) that is in the nanomolar or the picomolarrange.

The phrase “functional fragment” includes fragments of epitope-bindingproteins that can be expressed, secreted, and specifically bind to anepitope with a K_(D) in the micromolar, nanomolar, or picomolar range.Specific recognition includes having a K_(D) that is at least in themicromolar range, the nanomolar range, or the picomolar range.

The term “germline” in reference to an immunoglobulin nucleic acidsequence includes a nucleic acid sequence that can be passed to progeny.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. A heavy chain variable domain is encoded by a variable region genesequence, which generally comprises V_(H), D_(H), and J_(H) segmentsderived from a repertoire of V_(H), D_(H), and J_(H) segments present inthe germline. Sequences, locations and nomenclature for V, D, and Jheavy chain segments for various organisms can be found in IMGTdatabase, on the website of the International Immunogenetics InformationSystem.

The term “identity” when used in connection with sequence, includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences, but in the case of a light chainconstant domain, the length should contain sequence of sufficient lengthto fold into a light chain constant domain that is capable ofself-association to form a canonical light chain constant domain, e.g.,capable of forming two beta sheets comprising beta strands and capableof interacting with at least one C_(H)1 domain of a human or a mouse. Inthe case of a C_(H)1 domain, the length of sequence should containsequence of sufficient length to fold into a C_(H)1 domain that iscapable of forming two beta sheets comprising beta strands and capableof interacting with at least one light chain constant domain of a mouseor a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappaand lambda light chains and a VpreB, as well as surrogate light chains.Light chain variable domains typically include three light chain CDRsand four framework (FR) regions, unless otherwise specified. Generally,a full-length light chain includes, from amino terminus to carboxylterminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. Alight chain variable domain is encoded by a light chain variable regiongene sequence, which generally comprises V_(L) and J_(L) segments,derived from a repertoire of V and J segments present in the germline.Sequences, locations and nomenclature for V and J light chain segmentsfor various organisms can be found in IMGT database, www.imgt.org. Lightchains include those, e.g., that do not selectively bind either a firstor a second epitope selectively bound by the epitope-binding protein inwhich they appear. Light chains also include those that bind andrecognize, or assist the heavy chain with binding and recognizing, oneor more epitopes selectively bound by the epitope-binding protein inwhich they appear. Common or universal light chains include thosederived from a human Vκ1-39Jκ5 gene or a human Vκ3-20Jκ1 gene, andinclude somatically mutated (e.g., affinity matured) versions of thesame.

The phrase “micromolar range” is intended to mean 1-999 micromolar; thephrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase“picomolar range” is intended to mean 1-999 picomolar.

The phrase “somatically mutated” includes reference to a nucleic acidsequence from a B cell that has undergone class-switching, wherein thenucleic acid sequence of an immunoglobulin variable region (e.g.,nucleotide sequence encoding a heavy chain variable domain or includinga heavy chain CDR or FR sequence) in the class-switched B cell is notidentical to the nucleic acid sequence in the B cell prior toclass-switching, such as, for example, a difference in a CDR orframework nucleic acid sequence between a B cell that has not undergoneclass-switching and a B cell that has undergone class-switching.“Somatically mutated” includes reference to nucleic acid sequences fromaffinity-matured B cells that are not identical to correspondingimmunoglobulin variable region sequences in B cells that are notaffinity-matured (i.e., sequences in the genome of germline cells). Thephrase “somatically mutated” also includes reference to animmunoglobulin variable region nucleic acid sequence from a B cell afterexposure of the B cell to an epitope of interest, wherein the nucleicacid sequence differs from the corresponding nucleic acid sequence priorto exposure of the B cell to the epitope of interest. The phrase“somatically mutated” refers to sequences from antibodies that have beengenerated in an animal, e.g., a mouse having human immunoglobulinvariable region nucleic acid sequences, in response to an immunogenchallenge, and that result from the selection processes inherentlyoperative in such an animal.

The term “unrearranged,” with reference to a nucleic acid sequence,includes nucleic acid sequences that exist in the germline of an animalcell.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “operably linked” refers to a relationship wherein thecomponents operably linked function in their intended manner. In oneinstance, a nucleic acid sequence encoding a protein may be operablylinked to regulatory sequences (e.g., promoter, enhancer, silencersequence, etc.) so as to retain proper transcriptional regulation. Inone instance, a nucleic acid sequence of an immunoglobulin variableregion (or V(D)J segments) may be operably linked to a nucleic acidsequence of an immunoglobulin constant region so as to allow properrecombination between the sequences into an immunoglobulin heavy orlight chain sequence.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence.

“Functional” as used herein, e.g., in reference to a functionalpolypeptide, includes a polypeptide that retains at least one biologicalactivity normally associated with the native protein. In anotherinstance, a functional immunoglobulin gene segment may include avariable gene segment that is capable of productive rearrangement togenerate a rearranged immunoglobulin gene sequence.

“Neutral pH” includes pH between about 7.0 and about 8.0, e.g., pHbetween about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4,e.g., physiological pH. “Acidic pH” includes pH of 6.0 or lower, e.g.,pH between about 5.0 and about 6.0, pH between about 5.75 and about 6.0,e.g., pH of endosomal or lysosomal compartments.

Engineered Histidine Residues in Immunoglobulin Light Chain Genes

The inventors have discovered that non-human animals that expressantibodies that are capable of binding to an antigen in a pH dependentmanner can be made by making modifications of an immunoglobulin lightchain variable region at one or more positions along the sequence of thelight chain. Methods of making modifications in the germline of anon-human animal so that the animal would express histidines in CDRs ofantibodies are described. In particular, methods for makingmodifications in an immunoglobulin light chain variable sequence in thegermline of the mouse are described. Variable region sequence, e.g., oflight chains, typically show somatic hypermutation along the variableregion sequence, and, in some cases, such mutations can result in asubstitution of histidine residues (see, e.g., FIG. 1). Such mutationscan even occur in complementary determining regions (CDRs), which arethe regions of variable domains responsible for antigen binding. In somecases, such mutations can result in antibodies that display pH-dependentantigen binding, e.g., reduced antigen binding at an acidic pH ascompared to antigen binding at a neutral pH. Such pH-dependent antigenbinding is desired because it may enable the antibody to bind to theantigen outside the cell, and, when internalized into an endosome,release the antigen and recycle back to the surface to bind anotherantigen, avoiding target-mediated clearance. Approaches for introducinghistidine residues to achieve this effect by using a random his-scanningmutagenesis to engineer pH-dependent binding properties in anti-IL-6Rantibodies have been reported (US 2011/0111406 A1). However, randommutagenesis of antibody residues may result in decreased affinity ofantibody to the antigen. A non-human animal genetically modified toexpress a histidine substitution in antibody sequence enables generationof high-affinity antibodies in response to an antigen of interest that,due to histidine modification(s), would also display pH-dependentantigen binding.

Thus, in various embodiments, provided herein is a genetically modifiednon-human animal (e.g., rodent, e.g., a mouse or a rat) that comprisesin its genome, e.g., in its germline, a human immunoglobulin light chainvariable region sequence comprising modifications that result in theanimal expressing antibodies capable of binding to antigens in apH-dependent manner. In one embodiment, the non-human animal comprisesmodifications in the human immunoglobulin light chain variable regionsequence (e.g., V_(L) and/or J_(L) segment sequence) that comprisesubstitutions in at least one non-histidine codon with a histidine codon(in some cases, also may be referred to as “histidine substitution,”“histidine codon substitution,” or the like). In one embodiment, theanimal comprises at least one substitution of a non-histidine codon witha histidine codon in a nucleotide sequence of a complementarydetermining region (CDR; e.g., CDR1, CDR2, and/or CDR3) of a humanimmunoglobulin light chain. In one embodiment, the substitution is in aCDR3 codon. In one embodiment, the light chain is a κ light chain. Inone embodiment, the animal expresses an immunoglobulin light chain,e.g., a light chain CDR, e.g., a light chain CDR3, comprising asubstitution of at least one amino acid with a histidine. In anotherembodiment, the light chain is a λ light chain. In yet anotherembodiment, the mouse comprises a substitution of at least onenon-histidine codon with a histidine codon in both κ and λ light chains.

Histidine residue is encoded by two different codons, CAT and CAC(deoxyribonucleic acid residues). Thus, a non-histidine codon may besubstituted with a CAT or a CAC. The substitution is engineered in acodon that in its germline configuration (i.e., non-somatically mutatedstate) does not encode a histidine residue.

In one embodiment a light chain is a universal light chain (also termeda common light chain). As described in U.S. patent application Ser. Nos.13/022,759, 13/093,156, 13/412,936 and 13/488,628 (U.S. ApplicationPublication Nos. 2011/0195454, 2012/0021409, 2012/0192300 and2013/0045492, all incorporated herein by reference), a non-human animal(e.g., a mouse) that selects a common light chain for a plurality ofheavy chains has a practical utility. In various embodiments, antibodiesexpressed in a non-human animal comprising only a common light chainwill have heavy chains that can associate and express with an identicalor substantially identical light chain. This is particularly useful inmaking bispecific antibodies. For example, such an animal can beimmunized with a first immunogen to generate a B cell that expresses anantibody that specifically binds a first epitope. The animal (or ananimal genetically the same) can be immunized with a second immunogen togenerate a B cell that expresses an antibody that specifically binds thesecond epitope. Variable heavy chain regions can be cloned from the Bcells and expressed with the same heavy chain constant region and thesame light chain (e.g., a common light chain) in a cell to make abispecific antibody, wherein the heavy chain component of the bispecificantibody has been selected by an animal to associate and express withthe same light chain component. In various embodiments described, thevariable regions of the genetically engineered mice are human variableregions.

Thus, a mouse was engineered that is capable of generatingimmunoglobulin light chains that will suitably pair with a ratherdiverse family of heavy chains, including heavy chains whose humanvariable regions depart from germline sequences, e.g., affinity maturedor somatically mutated variable regions. In various embodiments, themouse is devised to pair human light chain variable domains with humanheavy chain variable domains that comprise somatic mutations, thusenabling a route to high affinity binding proteins suitable for use ashuman therapeutics.

The genetically engineered mouse, through the long and complex processof antibody selection within an organism, makes biologically appropriatechoices in pairing a diverse collection of human heavy chain variabledomains with a limited number of human light chain options. In order toachieve this, the mouse is engineered to present a limited number ofhuman light chain variable domain options in conjunction with a widediversity of human heavy chain variable domain options. Upon challengewith an immunogen, the mouse maximizes the number of solutions in itsrepertoire to develop an antibody to the immunogen, limited largely orsolely by the number or light chain options in its repertoire. Invarious embodiments, this includes allowing the mouse to achievesuitable and compatible somatic mutations of the light chain variabledomain that will nonetheless be compatible with a relatively largevariety of human heavy chain variable domains, including in particularsomatically mutated human heavy chain variable domains.

The engineered common light chain mouse described in U.S. ApplicationPublication Nos. 2011/0195454, 2012/0021409, 2012/0192300 and2013/0045492 comprised nucleic acid sequence encoding a limitedrepertoire of light chain options, e.g., common or universal light chain“ULC” that comprised no more than two V_(L) segments or a singlerearranged human immunoglobulin light chain variable region sequence. Toachieve such limited repertoire, a mouse was engineered to rendernonfunctional or substantially nonfunctional its ability to make, orrearrange, a native mouse light chain variable domain. In one aspect,this was achieved, e.g., by deleting the mouse's light chain variableregion gene segments. As previously described, the endogenous mouselocus can then be modified by exogenous suitable human light chainvariable region gene segments of choice, operably linked to theendogenous mouse light chain constant domain, in a manner such that theexogenous human variable region gene segments can combine with theendogenous mouse light chain constant region gene and form a rearrangedreverse chimeric light chain gene (human variable, mouse constant). Invarious embodiments, the light chain variable region is capable of beingsomatically mutated. In various embodiments, to maximize ability of thelight chain variable region to acquire somatic mutations, theappropriate enhancer(s) is retained in the mouse. In one aspect, inmodifying a mouse κ light chain locus to replace endogenous mouse κlight chain gene segments with human κ light chain gene segments, themouse κ intronic enhancer and mouse κ 3′ enhancer are functionallymaintained, or undisrupted.

Thus, provided was a genetically engineered mouse that expresses alimited repertoire of reverse chimeric (human variable, mouse constant)light chains associated with a diversity of reverse chimeric (humanvariable, mouse constant) heavy chains. In various embodiments, theendogenous mouse κ light chain gene segments are deleted and replacedwith a single (or two) rearranged human light chain region, operablylinked to the endogenous mouse Cκ gene. In embodiments for maximizingsomatic hypermutation of the rearranged human light chain region, themouse κ intronic enhancer and the mouse κ 3′ enhancer are maintained. Invarious embodiments, the mouse also comprises a nonfunctional λ lightchain locus, or a deletion thereof or a deletion that renders the locusunable to make a λ light chain.

The universal light chain mouse generated antibodies in response tovarious antigens that were capable of utilizing a diverse repertoire ofheavy chain variable region sequences, comprising a diverse repertoireof V_(H), D_(H), and J_(H) segments. Antibodies generated in suchgenetically engineered ULC mouse are useful for designing bispecifictherapeutic antibodies; however, as with any other antibody, eachbispecific antibody may only bind to one target during its lifetime inthe plasma; the antibody is internalized into an endosome and targetedfor lysosomal degradation. Studies have shown that MHC-class-I-like Fcγreceptor FcRn is capable of rescuing immunoglobulins from lysosomaldegradation by recycling it back to the cell surface from the sortingendosome. Simister and Mostov (1989) An Fc receptor structurally relatedto MHC class I antigens. Nature 337: 184-87. As explained above, toimprove efficiency of antibody recycling, further modifications toantibody sequences, e.g., modifications that result in decreased antigenbinding at acidic pH (e.g., pH of the endosome), while retainingantibody-antigen affinity and specificity at neutral pH (e.g.,physiological pH) are beneficial. The non-human animals describedherein, wherein histidine residues are substituted for non-histidineresidues in the a universal light chain sequence are beneficial becausethey are capable of producing high-affinity antibodies based onuniversal light chain format that also display pH-dependent binding,e.g., display reduced binding to the antigen at acidic versus neutralpH.

Thus, in one embodiment, provided herein is a non-human animal (e.g., arodent, e.g., a mouse or a rat) that comprises in its genome, e.g., inits germline, a limited repertoire of human light chain variableregions, or a single human light chain variable region, from a limitedrepertoire of human light chain variable gene segments, wherein thehuman light chain variable region(s) comprise at least one substitutionof a non-histidine codon for a histidine codon. In some embodiments,provided non-human animals are genetically engineered to include asingle unrearranged human light chain variable region gene segment (ortwo human light chain variable region gene segments) that rearranges toform a rearranged human light chain variable region gene (or tworearranged light chain variable region genes) that expresses a singlelight chain (or that express either or both of two light chains),wherein the light chain variable region gene(s) comprise a substitutionof at least one non-histidine codon with a histidine codon. Therearranged human light chain variable domains encoded by thesehistidine-substituted light chain variable region gene(s) are capable ofpairing with a plurality of affinity-matured human heavy chains selectedby the animals, wherein the heavy chain variable regions specificallybind different epitopes. In various embodiments, the at least onesubstitution of a non-histidine residue with a histidine residue resultsin a rearranged human light chain that, when expressed with a cognateheavy chain, binds to its antigen in a pH-dependent manner.

Genetically engineered animals are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region gene sequences, wherein the variable region genesequences comprise at least one substitution of a non-histidine codonwith a histidine codon. In some embodiments, provided animals aregenetically engineered to include a single V/J human light chainsequence (or two V/J sequences) that comprises a substitution of atleast one non-histidine codon with a histidine codon and expresses avariable region of a single light chain (or that express either or bothof two variable regions). In one aspect, a light chain comprising thevariable sequence is capable of pairing with a plurality ofaffinity-matured human heavy chains clonally selected by the animal,wherein the heavy chain variable regions specifically bind differentepitopes. In one embodiment, the antibody binds to its antigen(s) in apH-dependent manner. In one embodiment, the single V/J human light chainsequence is selected from Vκ1-39Jκ5 and Vκ3-20Jκ1. In one embodiment,the two V/J sequences are Vκ1-39Jκ5 and Vκ3-20Jκ1. In one embodiment,the Vκ1-39Jκ5 and Vκ3-20Jκ1 sequences are rearranged V/J sequences.

In one aspect, a genetically modified non-human animal is provided thatcomprises a single human immunoglobulin light chain V_(L) gene segmentthat is capable of rearranging with a human J_(L) gene segment (selectedfrom one or a plurality of J_(L) segments) and encoding a human variabledomain of an immunoglobulin light chain, wherein the single humanimmunoglobulin light chain V_(L) gene segment and/or human J_(L) genesegment comprise a substitution of at least one non-histidine codon witha histidine codon. In another aspect, a genetically modified mouse isprovided that comprises no more than two human V_(L) gene segments, eachof which is capable of rearranging with a human J_(L) gene segment(selected from one or a plurality of J_(L) segments) and encoding ahuman variable domain of an immunoglobulin light chain, wherein each ofthe no more than two V_(L) gene segments and/or the J_(L) gene segmentcomprise a substitution of at least one non-histidine residue with ahistidine residue.

Also provided herein is a genetically modified non-human animal thatcomprises in its genome, e.g., in its germline, a single rearrangedhuman immunoglobulin light chain variable region sequence comprisinghuman V_(L) and J_(L) sequences wherein the single rearranged humanimmunoglobulin light chain variable region comprises a substitution ofat least one non-histidine codon with a histidine codon. In one aspect,the single rearranged human immunoglobulin light chain variable regionsequence is derived from human germline V_(L) and J_(L) gene sequences,but for the histidine substitution(s). In one embodiment, the humanimmunoglobulin light chain is a human immunoglobulin K chain. Thus, inone embodiment, the human V_(L) gene sequence is selected from Vκ1-39and Vκ3-20. In one embodiment, the single rearranged humanimmunoglobulin light chain variable region sequence comprises rearrangedVκ1-39/J or Vκ3-20/J sequence. In one embodiment, the human J_(L) genesequence is selected from Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In one embodimentthe human J_(L) sequence is selected from Jκ1 and Jκ5. In oneembodiment, the single rearranged human immunoglobulin light chainvariable region sequence is selected from Vκ1-39Jκ5 and Vκ3-20Jκ1 (e.g.,but for the histidine substitution(s)). In an alternative embodiment,the human immunoglobulin light chain is a human λ chain.

In one embodiment, the substitution of at least one non-histidine codonfor a histidine codon is in the nucleotide sequence encoding acomplementary determining region (CDR) of the light chain variabledomain. In one embodiment, the substitution of at least onenon-histidine codon for a histidine codon is in the nucleotide sequenceencoding CDR1, CDR2 or CDR3 of the light chain variable domain. In onespecific embodiment, the substitution is in the nucleotide sequenceencoding CDR3.

In one aspect, the substitution is of at least one non-histidine codonfor a histidine codon in the CDR3 codon of the human light chainvariable region gene sequence. In one embodiment, the substitution is ofone, two, three, four, or more CDR3 codons. In the embodiment whereinthe single rearranged human immunoglobulin light chain variable regionis a Vκ1-39Jκ5 variable region, the replacement of at least onenon-histidine codon with a histidine codon comprises a replacement at aposition in the immunoglobulin light chain gene sequence encoding CDR3designed to express a histidine at position selected from 105, 106, 108,111, and a combination thereof. In one embodiment, the replacement isdesigned to express histidines at positions 105 and 106. In oneembodiment, the replacement is designed to express histidines atpositions 105 and 111. In one embodiment, the replacement is designed toexpress histidines at positions 105 and 108. In one embodiment, thereplacement is designed to express histidines at positions 105, 108 and111. In one embodiment, the replacement is designed to expresshistidines at positions 105, 106, and 108. In one embodiment, thereplacement is designed to express histidines at positions 106 and 108.In one embodiment, the replacement is designed to express histidines atpositions 106 and 111. In one embodiment, the replacement is designed toexpress histidines at positions 108 and 111. In one embodiment, thereplacement is designed to express histidines at positions 106, 108, and111. In yet another embodiment, the replacement is designed to expresshistidines at positions 106, 108 and 111. In one embodiment, thereplacement is designed to express histidines at positions 105, 106, and111. In one embodiment, the replacement is designed to expresshistidines at positions 105, 106, 108, and 111. The nucleic acid andamino acid sequences of the histidine-substituted CDR3 regions aredepicted in sequence alignment of FIG. 2 and set forth in SEQ ID NOs:4-33. Wild type CDR3 nucleic acid and amino acid sequences (depicted inFIG. 2) are set forth in SEQ ID NOs:2 and 3, respectively.

In the embodiment wherein the single rearranged human immunoglobulinlight chain variable region is a Vκ3-20Jκ1 variable region, thereplacement of at least one non-histidine codon with a histidine codoncomprises a replacement at a position in the immunoglobulin light chaingene sequence encoding CDR3 region that is designed to express ahistidine at position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, the replacement is designed toexpress histidines at positions 105 and 106. In one embodiment, thereplacement is designed to express histidines at positions 105 and 107.In one embodiment, the replacement is designed to express histidines atpositions 105 and 109. In one embodiment, the replacement is designed toexpress histidines at positions 106 and 107. In one embodiment, thereplacement is designed to express histidines at positions 106 and 109.In one embodiment, the replacement is designed to express histidines atpositions 107 and 109. In one embodiment, the replacement is designed toexpress histidines at positions 105, 106, and 107. In one embodiment,the replacement is designed to express histidines at positions 105, 107,and 109. In one embodiment, the replacement is designed to expresshistidines at positions 106, 108, and 111. In one embodiment, thereplacement is designed to express histidines at positions 105, 106 and109. In another embodiment, the replacement is designed to expresshistidines at positions 105, 106, 107, and 109. The nucleic acid andamino acid sequences of exemplary histidine-substituted CDR3 regions aredepicted in sequence alignment of FIG. 12 and set forth in SEQ ID NOs:76-79. Wild type CDR3 nucleic acid and amino acid sequences (depicted inFIG. 12) are set forth in SEQ ID NOs:74 and 75, respectively.

Amino acid positions (105, 106, etc.) are based on a unique numberingdescribed in Lefranc et al. (2003) Dev. Comp. Immunol. 27:55-77, and canalso be viewed on www.imgt.org.

In one embodiment, the human V_(L) gene segment is operably linked to ahuman or non-human leader sequence. In one embodiment, the leadersequence is a non-human leader sequence. In a specific embodiment, thenon-human leader sequence is a mouse Vκ3-7 leader sequence. In aspecific embodiment, the leader sequence is operably linked to anunrearranged human V_(L) gene segment. In a specific embodiment, theleader sequence is operably linked to a rearranged human V_(L)/J_(L)sequence. Thus, in one specific embodiment, the single rearrangedVκ1-39/Jκ5 or Vκ3-20/Jκ1 variable region gene sequence comprising atleast one histidine substitution is operably linked to a mouse Vκ3-7leader sequence.

In one embodiment, the V_(L) gene segment is operably linked to animmunoglobulin promoter sequence. In one embodiment, the promotersequence is a human promoter sequence. In a specific embodiment, thehuman immunoglobulin promoter is a human Vκ3-15 promoter. In a specificembodiment, the promoter is operably linked to an unrearranged humanV_(L) gene segment. In a specific embodiment, the promoter is operablylinked to a rearranged human V_(L)/J_(L) sequence. Thus, in one specificembodiment, the single rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 variableregion gene sequence comprising at least one histidine substitution isoperably linked to the human Vκ3-15 promoter.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with ahuman V_(L) gene segment that rearranges with a human J_(L) segment andencodes a variable domain of a reverse chimeric light chain comprisingan endogenous non-human light chain constant region (C_(L)). In aspecific embodiment, the V_(L) and J_(L) gene segments are at thenon-human Vκ locus, and the non-human C_(L) is a non-human Cκ (e.g.,mouse Cκ). In one specific embodiment, the variable region sequence isoperably linked to the non-human constant region sequence, e.g., thenon-human Cκ gene sequence.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with arearranged human variable region sequence (V_(L)/J_(L) sequence) andencodes a variable domain of a reverse chimeric light chain comprisingan endogenous non-human light chain constant region (C_(L)). In aspecific embodiment, the rearranged human V_(L)/J_(L) sequence is at thenon-human kappa (κ) locus, and the non-human C_(L) is a non-human Cκ. Inone specific embodiment, the rearranged human variable region sequenceis operably linked to the non-human immunoglobulin light chain constantregion sequence, e.g., the non-human Cκ gene sequence. In oneembodiment, the non-human immunoglobulin light chain constant regionsequence is an endogenous non-human sequence. In one embodiment, thenon-human animal is a mouse and the Cκ gene sequence is a mouse Cκ genesequence. In one embodiment, the rearranged human immunoglobulin lightchain variable region sequence comprising a substitution of at least onenon-histidine codon with a histidine codon is at the endogenousnon-human (e.g., mouse) immunoglobulin light chain locus (κ locus).Exemplary embodiments of the locus are presented in FIGS. 8C, 8E, 14C,and 14D.

In one embodiment, the genetically modified non-human animal is a mouse,and the variable region locus of the mouse is a κ light chain locus, andthe κ light chain locus comprises a mouse κ intronic enhancer, a mouse κ3′ enhancer, or both an intronic enhancer and a 3′ enhancer.

In one embodiment, the non-human animal (e.g., a rodent, e.g., a rat ora mouse) comprises a nonfunctional immunoglobulin lambda (λ) light chainlocus. In a specific embodiment, the λ light chain locus comprises adeletion of one or more sequences of the locus, wherein the one or moredeletions renders the λ light chain locus incapable of rearranging toform a light chain gene. In another embodiment, all or substantially allof the V_(L) gene segments of the λ light chain locus are deleted. Inone embodiment, the non-human animal (e.g., rodent, e.g. mouse or rat)comprises a rearranged human immunoglobulin light chain variable regionsequence comprising a substitution of at least one non-histidine codonwith a histidine codon, and lacks a functional unrearrangedimmunoglobulin light chain variable region, e.g., endogenousunrearranged light chain variable region. In one embodiment, therearranged, histidine-substituted human immunoglobulin light chainvariable region gene sequence replaces endogenous unrearrangedimmunoglobulin light chain variable region gene sequence.

In one embodiment, the animal makes a light chain that comprises asomatically mutated variable domain derived from a human variable regionsequence that comprises a substitution of at least one non-histidinecodon with a histidine codon. In one embodiment, the light chaincomprises a somatically mutated variable domain derived from a humanvariable region sequence that comprises a substitution of at least onenon-histidine codon with a histidine codon, and a non-human Cκ region.In one embodiment, the non-human animal does not express a λ lightchain.

One skilled in the art would appreciate that although substitution(s) ofat least one non-histidine residue with a histidine residue isgenetically engineered into the human immunoglobulin light chainvariable region, due to somatic hypermutations, not all antibodies thatare generated in the genetically modified non-human animal will harborthat histidine residue(s) at engineered position(s). However, generationof a wide repertoire of antibodies in the non-human animal will allow toselect for in vivo generated antigen-specific antibodies that displayhigh affinity for an antigen of interest while retaining histidinemodifications introduced into the germline and, preferably, exhibitingpH-dependent antigen binding.

Thus, in one embodiment, the animal retains at least one histidine aminoacid introduced by substitution of at least one non-histidine codon witha histidine codon in its variable region gene. In one embodiment, theanimal retains all or substantially all histidine substitutions in itssomatically mutated light chain variable domain that were introducedinto its variable region gene.

In one embodiment, the genetically modified non-human animal describedherein also comprises in its genome, e.g., in its germline, anunrearranged immunoglobulin heavy chain variable region comprisingV_(H), D_(H), and J_(H) gene segment sequences. In one embodiment, theV_(H), D_(H), and J_(H) gene segment sequences are human V_(H), D_(H),and J_(H) gene segment sequences, and the unrearranged immunoglobulinheavy chain variable region is a human heavy chain variable region. Inone embodiment, the human V_(H), D_(H), and J_(H) gene segment sequencesare operably linked to non-human heavy chain constant region sequence.In one embodiment, the non-human heavy chain constant region sequence isan endogenous non-human heavy chain constant region sequence. In oneembodiment, the human heavy chain gene segment sequences are at theendogenous non-human immunoglobulin heavy chain locus. In oneembodiment, the human immunoglobulin heavy chain variable regionsequence comprised in a non-human animal also comprises a substitutionof at least one non-histidine codon for a histidine codon.

In one embodiment, the non-human animal described herein expresses animmunoglobulin light chain that comprises a non-human light chainconstant region sequence. In one embodiment, the non-human animalexpresses an immunoglobulin light chain that comprises a human lightchain constant region sequence.

In one embodiment, the non-human animal described herein expresses animmunoglobulin heavy chain that comprises a non-human sequence selectedfrom a C_(H)1 sequence, a hinge sequence, a C_(H)2 sequence, a C_(H)3sequence, and a combination thereof.

In one embodiment, the non-human animal expresses an immunoglobulinheavy chain that comprises a human sequence selected from a C_(H)1sequence, a hinge sequence, a C_(H)2 sequence, a C_(H)3 sequence, and acombination thereof.

In the embodiment where the animal comprises a single rearrangedimmunoglobulin light chain variable region comprising a substitution ofat least one non-histidine codon with a histidine codon, the rearrangedimmunoglobulin light chain sequence in the germline of the animal is atan endogenous non-human immunoglobulin light chain locus. In a specificembodiment, the rearranged immunoglobulin light chain sequencecomprising a substitution of at least one non-histidine codon with ahistidine codon in the germline of the animal replaces all orsubstantially all endogenous non-human light chain V and J segmentsequences at the endogenous non-human immunoglobulin light chain locus.

In one embodiment, the non-human animal comprises a replacement ofendogenous V_(H) gene segments with one or more human V_(H) genesegments, wherein the human V_(H) gene segments are operably linked to anon-human C_(H) region gene, such that the non-human animal rearrangesthe human V_(H) gene segments and expresses a reverse chimericimmunoglobulin heavy chain that comprises a human V_(H) domain and anon-human C_(H). In one embodiment, 90-100% of unrearranged non-humanV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially all(e.g., 90-100%) of the endogenous non-human V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In oneembodiment, the replacement is with at least 19, at least 39, or atleast 80 or 81 unrearranged human V_(H) gene segments. In oneembodiment, the replacement is with at least 12 functional unrearrangedhuman V_(H) gene segments, at least 25 functional unrearranged humanV_(H) gene segments, or at least 43 functional unrearranged human V_(H)gene segments. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with at least oneunrearranged human D_(H) segment and at least one unrearranged humanJ_(H) segment. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with allunrearranged human D_(H) segments and all unrearranged human J_(H)segments.

A non-human animal, e.g., a mouse, comprising in its genome, e.g., inits germline, a limited repertoire of human immunoglobulin light chainvariable regions, e.g., a single rearranged human immunoglobulin lightchain variable region (e.g., Vκ1-39/Jκ5 or Vκ3-20/Jκ1), with asubstitution of at least one non-histidine codon with a histidine codonand a diverse repertoire of unrearranged human V_(H), D_(H), and J_(H)segments is capable of generating antigen binding proteins encoded byheavy chain variable region sequences derived from various permutationsof unrearranged human V_(H), D_(H), and J_(H) segments, wherein theV_(H), D_(H), and J_(H) segments present in the heavy chain variablesequences are derived from all or substantially all functional humanV_(H), D_(H), and J_(H) segments present in the genome of the animal.Various available possibilities for heavy chain variable domainsequences expressed in the cells, e.g., B cells, of the geneticallymodified animals described herein (i.e., derived from combinations ofvarious functional human V, D, and J segments) are described in U.S.Application Publication Nos. 2011/0195454, 2012/0021409, 2012/0192300and 2013/0045492, all incorporated herein by reference. In variousembodiments, the rearranged human immunoglobulin light chain variableregion sequence comprising substitution(s) of at least one non-histidinecodon with a histidine codon and the unrearranged human immunoglobulinheavy chain variable region sequence are comprised in the germline ofthe non-human animal.

In one embodiment, the non-human animal comprises one copy of one orboth of the rearranged human immunoglobulin light chain variable regionsequence comprising substitution(s) of at least one non-histidine codonwith a histidine codon and the unrearranged human immunoglobulin heavychain variable region sequence. In another embodiment, the non-humananimal comprises two copies of one or both of the rearranged humanimmunoglobulin light chain variable region sequence comprisingsubstitution(s) of at least one non-histidine codon with a histidinecodon and the unrearranged human immunoglobulin heavy chain variableregion sequence. Thus, the non-human animal may be homozygous orheterozygous for one or both the rearranged human immunoglobulin lightchain variable region sequence comprising substitution(s) of at leastone non-histidine codon with a histidine codon and the unrearrangedhuman immunoglobulin heavy chain variable region sequence.

In addition to genetically modified non-human animals comprising intheir genome an immunoglobulin light chain variable region gene sequence(e.g., a single rearranged immunoglobulin light chain variable regiongene sequence) comprising substitution of at least one non-histidinecodon with a histidine codon (e.g., in CDR3 of the light chain), alsoprovided herein are genetically modified non-human animals comprising animmunoglobulin light chain variable region gene sequence with one ormore additions/insertions of histidine codon(s), such that the expressedvariable domain comprises an additional amino acid(s) which, if notsubject to somatic hypermutation, is a histidine.

The genetically modified non-human animal comprising a humanimmunoglobulin light chain variable region gene sequence with asubstitution of at least one non-histidine codon with a histidine codondescribed herein may be selected from a group consisting of a mouse,rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat,chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). Forthe non-human animals where suitable genetically modifiable ES cells arenot readily available, methods distinct from those described herein areemployed to make a non-human animal comprising the genetic modification.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 12951/SvIm),129S2, 129S4, 129S5, 12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the genetically modified non-human animal is arodent. In one embodiment, the genetically modified non-human animal isa rat or a mouse. In one embodiment, the animal is a mouse. Thus, in oneembodiment, provided herein is a genetically modified mouse comprisingin its genome, e.g., in its germline, a single rearranged humanimmunoglobulin light chain variable region comprising human V_(L) andJ_(L) gene sequences, wherein the single rearranged human immunoglobulinlight chain variable region comprises a substitution of at leastnon-histidine codon with a histidine codon. In one embodiment, the mouselacks a functional unrearranged immunoglobulin light chain variableregion (e.g., lacks functional unrearranged V and J gene segmentsequences). In one embodiment, the rearranged human immunoglobulin lightchain variable region with histidine codon substitution(s) is Vκ1-39/JKor Vκ3-20/JK variable region. In one embodiment the J segment sequenceis selected from Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In one embodiment the Jsegment sequence is Jκ1 or Jκ5. In one embodiment, the substitution ofat least one non-histidine codon with a histidine codon is in thenucleotide sequence encoding a CDR3 region. In one embodiment, whereinthe rearranged variable region sequence is Vκ1-39/Jκ5 sequence, thehistidine substitution(s) is designed to express at a position selectedfrom 105, 106, 108, 111, and a combination thereof. In anotherembodiment, wherein the rearranged variable region sequence isVκ3-20/Jκ1 sequence, the histidine substitution(s) is designed toexpress at a position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, the rearranged immunoglobulinlight chain variable region with substituted histidine codon(s) isoperably linked to an endogenous mouse immunoglobulin constant regiongene sequence (e.g., Cκ gene sequence). In one embodiment, the mousefurther comprises in its genome, e.g., in its germline, an unrearrangedimmunoglobulin heavy chain variable region comprising human V_(H),D_(H), and J_(H) segments. In one embodiment, human V_(H), D_(H), andJ_(H) segments are operably linked to an endogenous mouse immunoglobulinheavy chain constant region gene sequence. In various embodiments, therearranged human immunoglobulin light chain variable region sequencecomprising substitution(s) of at least one non-histidine codon with ahistidine codon and the unrearranged human immunoglobulin heavy chainvariable region sequence are comprised in the germline of the mouse.

Also provided herein are targeting vectors for generating geneticallymodified non-human animals, e.g., mice, described herein. In one aspect,provided is a targeting vector comprising, from 5′ to 3′ intranscriptional direction with reference to the sequences of the 5′ and3′ mouse homology arms of the vector, a 5′ mouse homology arm, a humanor mouse immunoglobulin promoter, a human or mouse leader sequence, ahuman variable region selected from a rearranged human Vκ1-39Jκ5 or arearranged human Vκ3-20Jκ1 and comprising a substitution of at least onenon-histidine codon with a histidine codon, and a 3′ mouse homology arm.In one embodiment, the 5′ and 3′ homology arms target the vector to asequence 5′ with respect to an enhancer sequence that is present 5′ andproximal to the mouse Cκ gene. In another embodiment, the targetingvector comprises a 5′ mouse homology arm followed by a selectioncassette flanked by recombination sites, human or mouse immunoglobulinpromoter, human or mouse leader sequence, a human variable regionselected from a rearranged human Vκ1-39Jκ5 or a rearranged humanVκ3-20Jκ1 and comprising a substitution of at least one non-histidinecodon with a histidine codon, followed by the 3′ mouse homology arm thatcomprises mouse enhancers and constant region (Cκ) sequences.

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., ES cells) that haveintegrated the construct of interest. A number of suitable selectioncassettes are known in the art. Commonly, a selection cassette enablespositive selection in the presence of a particular antibiotic (e.g.,Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette may beflanked by recombination sites, which allow deletion of the selectioncassette upon treatment with recombinase enzymes. Commonly usedrecombination sites are loxP and Frt, recognized by Cre and Flp enzymes,respectively, but others are known in the art.

In one embodiment, the promoter is a human immunoglobulin variableregion gene segment promoter. In a specific embodiment, the promoter isa human Vκ3-15 promoter. In one embodiment, the leader sequence is amouse leader sequence. In a specific embodiment, the mouse leadersequence is a mouse Vκ3-7 leader sequence. Exemplary embodiments of thetargeting vectors are presented in FIGS. 8B and 14B.

In one aspect, a targeting vector is provided as described above, but inplace of the 5′ mouse homology arm the human or mouse promoter isflanked 5′ with a site-specific recombinase recognition site (SRRS), andin place of the 3′ mouse homology arm the human V_(L) region is flanked3′ with an SRRS.

Also provided herein are methods of making genetically modifiednon-human animals (e.g., rodents, e.g., mice or rats) described herein.In one aspect, the method for making a genetically modified non-humananimal described herein utilizes a targeting vector, made usingVELOCIGENE® technology, introducing the construct into ES cells, andintroducing targeted ES cell clones into a mouse embryo usingVELOCIMOUSE® technology, as described in the Examples. Histidinemodifications may be introduced into the targeting vector using avariety of molecular biology techniques, e.g., site directed mutagenesisor de novo DNA synthesis. Upon completion of gene targeting, ES cells ofgenetically modified non-human animals are screened to confirmsuccessful incorporation of exogenous nucleotide sequence of interest orexpression of exogenous polypeptide. Numerous techniques are known tothose skilled in the art, and include (but are not limited to) Southernblotting, long PCR, quantitative PCT (e.g., real-time PCR usingTAQMAN®), fluorescence in situ hybridization, Northern blotting, flowcytometry, Western analysis, immunocytochemistry, immunohistochemistry,etc. In one example, non-human animals (e.g., mice) bearing the geneticmodification of interest can be identified by screening for loss ofmouse allele and/or gain of human allele using a modification of alleleassay described in Valenzuela et al. (2003) High-throughput engineeringof the mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6):652-659. Other assays that identify a specificnucleotide or amino acid sequence in the genetically modified animalsare known to those skilled in the art.

Thus, in one embodiment, the method of generating genetically modifiednon-human animals comprises replacing an immunoglobulin light chainvariable region gene sequence in the animal with a human immunoglobulinlight chain variable region gene sequence (comprising human V_(L) andJ_(L) gene segments) wherein the human immunoglobulin variable regiongene sequence comprises a substitution of at least one non-histidinecodon with a histidine codon. In one embodiment, the substitution of atleast one non-histidine codon with a histidine codon is in thenucleotide sequence encoding a CDR region, e.g., a CDR3 region.

In one embodiment, the method of generating genetically modifiednon-human animals described herein comprises replacing an immunoglobulinlight chain variable region gene sequence in the animal with a singlerearranged human immunoglobulin light chain variable region genesequence comprising human V_(L) and J_(L) gene segment sequences,wherein the single rearranged human immunoglobulin variable region genesequence comprises a substitution of at least one non-histidine codonwith a histidine codon. In one embodiment, the substitution is in a CDRcodon. In one embodiment, the substitution is of one, two, three, four,or more CDR3 codon(s). In one embodiment, the single rearranged humanimmunoglobulin light chain variable region gene sequence is based on thehuman germline rearranged light chain variable region sequence selectedfrom Vκ1-39Jκ5 and Vκ3-20Jκ1. Thus, in one embodiment, where the singlerearranged human immunoglobulin light chain variable region genesequence is derived from Vκ1-39Jκ5, replacement of at least onenon-histidine codon with histidine codon is designed to express ahistidine at positions selected from 105, 106, 108, 111, and acombination thereof. In one embodiment, where the single rearrangedhuman immunoglobulin light chain variable region gene sequence isderived from Vκ3-20κ1, replacement of at least one non-histidine codonwith a histidine codon is designed to express a histidine at positionselected from 105, 106, 107, 109, and a combination thereof.

In another embodiment, the method of generating a non-human animaldescribed herein (i.e., comprising a genetically modified immunoglobulinlight chain locus described herein) comprises modifying a genome of anon-human animal to delete or render nonfunctional endogenousimmunoglobulin light chain V and J segments in an immunoglobulin lightchain locus, and placing in the genome a single rearranged human lightchain variable region gene sequence comprising a substitution of atleast one non-histidine codon with a histidine codon. In one embodiment,the method results in a genetically modified non-human animal thatcomprises a population of B cells enriched for antibodies exhibiting pHdependent binding to an antigen of interest.

In some embodiments, the methods of generating genetically modifiednon-human animals described herein comprise replacing an immunoglobulinlight chain variable region gene sequence with human immunoglobulinlight chain variable gene region sequence comprising substitution(s) ofat least one non-histidine codon with a histidine codon in the animalthat also comprises a replacement of endogenous non-human immunoglobulinheavy chain variable region gene sequence with a human immunoglobulinheavy chain variable region gene sequence comprising at least one ofeach or a repertoire of human V_(H), D_(H), and J_(H) sequences, asdescribed above. In one embodiment, in order to generate a non-humananimal comprising a replacement of endogenous immunoglobulin light chainvariable region gene sequence human light chain variable region genesequence comprising a substitution of at least one non-histidine codonwith a histidine codon and a replacement of endogenous non-humanimmunoglobulin heavy chain variable region gene sequence with a humanimmunoglobulin heavy chain variable region gene sequence, the animalwith replacement of light chain variable region gene sequence is bred toan animal with replacement of heavy chain variable region gene sequence.

Inventors presently provide genetically engineered non-human animals(e.g., rodents, e.g., rats or mice) that express antigen-bindingproteins, e.g., antibodies, that comprise a universal light chain, e.g.,a human universal light chain (e.g., a light chain derived from a singlerearranged human immunoglobulin light chain variable region) thatcomprises one or more histidine modifications, wherein theantigen-binding proteins exhibit a pH-dependent antigen binding of atarget antigen. The animals are genetically engineered to include alight chain CDR3 that comprises one or more histidine modifications. Invarious embodiments, the light chain CDR3 comprises two, three, or fouror more histidine residues in a cluster.

In one embodiment, provided herein is a genetically engineered non-humananimal (e.g., a mouse or a rat) that comprises a population of B cellscharacterized by enhanced presence of histidines in immunoglobulin lightchains, e.g., immunoglobulin variable domains, e.g., immunoglobulinCDRs, compared to a wild type animal. In one embodiment, enhancement ofhistidine presence is about 2 to 4 fold. In one embodiment, enhancementof histidines is about 2 to 10 fold.

In one embodiment, provided herein is a genetically engineered non-humananimal that comprises a population of antigen-specific antibodies thatexpress histidine residue(s) as a result of codon modifications in thelight chain variable region gene sequence, and display pH-dependentbinding of target antigen. In one embodiment, these animals comprise apopulation of B cells that are enriched for antibodies, e.g.,antigen-specific antibodies, that display pH-dependent bindingproperties (e.g., decreased dissociative half-life (t_(1/2))) at acidicpH vs neutral pH) as compared to a population of antigen-specificantibodies generated in animals that do not comprise a substitution ofat least one non-histidine codon with a histidine codon inimmunoglobulin light chain variable region described herein. In oneembodiment, the enrichment of antigen-specific antibodies displayingpH-dependent antigen binding properties generated in the geneticallyengineered animals described herein as compared to similar animals thatdo comprise histidine substitutions in light chain variable region isgreater than about 2 fold, e.g., greater than about 5 fold, e.g.,greater than about 10 fold. Thus, the genetically modified animals ofthe invention are enriched for antibodies with improved antibodyrecycling properties, which is desired in order to reducetarget-mediated clearance as well as to reduce the dose and/or dosingfrequency of a therapeutic antigen-binding protein developed based onsuch in vivo generated antibody format.

Thus, provided herein is an antigen-binding protein, generated ingenetically modified non-human animals described herein, wherein theantigen-binding protein displays pH-dependent antigen binding. In oneembodiment, the antigen-binding protein is an antibody, e.g.,antigen-specific antibody. In one embodiment, the antibody comprises alight chain which comprises a human light chain variable domain derivedfrom a rearrangement of human immunoglobulin light chain variable genesegments where at least one non-histidine codon was substituted for ahistidine codon in the germline gene sequence, and wherein the antibodyretains at least one histidine substitution in its expressed human lightchain variable domain. In one embodiment, the antibody comprises a lightchain which comprises a human light chain variable domain derived from asingle rearranged human light chain variable region gene sequence,wherein the single rearranged light chain variable region gene sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon, and wherein the antibody retains at least one histidinesubstitution in its expressed light chain variable domain. In oneembodiment, the antibody comprises a light chain derived from a humanVκ1-39Jκ5 or Vκ3-20Jκ1 rearrangement, wherein the human Vκ1-39Jκ5 orVκ3-20Jκ1 gene sequence comprises a substitution of at least onenon-histidine codon with a histidine codon, and wherein the antibodyretains at least one histidine substitution in its expressed light chainvariable domain. In some embodiments, the antibody retains all orsubstantially all histidine substitutions in its expressed light chainvariable domain. In one embodiment, the substitution is of threenon-histidine codons with three histidine codons in the nucleotidesequence encoding CDR3 of the light chain variable region gene sequence,and the antibody retains all three histidine substitutions in itsexpressed light chain variable domain. In one embodiment, thesubstitution is of four non-histidine codons with four histidine codonsin the nucleotide sequence encoding CDR3 of the light chain variableregion gene sequence, and the antibody retains three or four histidinesubstitutions in its expressed light chain variable domain.

In one embodiment, the light chain of the antibody further comprises anon-human light chain constant region amino acid sequence, e.g.,endogenous light chain constant region amino acid sequence. In addition,the antibody, e.g., antigen-specific antibody, generated in agenetically modified non-human animal described herein also comprises aheavy chain which comprises a human heavy chain variable domain derivedfrom a rearrangement of human heavy chain V, D, and J segments. Humanheavy chain V, D, and J segments may be selected from a repertoire ofhuman heavy chain segments present at the endogenous non-human heavychain locus, e.g., at least one functional V, at least one functional D,and at least one functional J segment, e.g., up to a complete repertoireof functional human V, D, and J segments. Exemplary possiblerearrangements of human heavy chain variable segments may be gleanedfrom a listing of functional human V, D, and J segments in IMGTdatabase, and from U.S. Application Publication Nos. 2011/0195454,2012/0021409, 2012/0192309, and 2013/0045492, incorporated herein byreference. Furthermore, in one embodiment, the heavy chain of theantibody comprises a non-human heavy chain constant region amino acidsequence, e.g., an endogenous non-human heavy chain constant regionamino acid sequence. In one embodiment, the non-human heavy chainconstant region comprises C_(H)1, hinge, C_(H)2, and C_(H)3 domains. Inone embodiment, the antibody is an IgG, IgE, IgD, IgM, or IgA isotype.

Thus, in one embodiment, provided herein is a binding protein generatedin the genetically modified non-human animals described herein, whereinthe binding protein comprises a reverse chimeric light chain comprising(a) a light chain variable domain derived from a human Vκ1-39Jκ5rearrangement comprising a substitution of at least one non-histidinecodon with a histidine codon, wherein the light chain retains at leastone histidine substitution in its expressed light chain variable domainand (b) a non-human, e.g., a mouse, light chain constant region aminoacid sequence, wherein the light chain is associated with a reversechimeric heavy chain comprising (a) a heavy chain variable domainderived from a rearrangement of human V, D, and J segments, wherein theV, D, and J segments are selected from a repertoire of human V, D, and Jsegments present in the animal, and (b) a non-human, e.g., mouse, heavychain constant region amino acid sequence. In one embodiment, therepertoire of human V, D, and J segments comprises at least onefunctional V, at least one functional D, and at least one functional Jsegment, e.g., up to a complete repertoire of functional human V, D, andJ segments. In one embodiment, the heavy and the light chain constantdomains are endogenous heavy and light chain constant regions. In oneembodiment, the heavy and light chain variable domains are somaticallymutated domains. In one embodiment, the somatically mutated light chaindomain retains at least one histidine substitution introduced into thegermline sequence. In some embodiments, the somatically mutated lightchain domain retains all or substantially all histidine substitutionsintroduced into the germline sequence. In one embodiment, theantigen-binding protein displays pH-dependent antigen bindingproperties.

In another embodiment, provided herein is a binding protein generated inthe genetically modified non-human animals described herein, wherein thebinding protein comprises a reverse chimeric light chain comprising (a)a light chain variable domain derived from a human Vκ3-20Jκ1rearrangement comprising a substitution of at least one non-histidinecodon with a histidine codon, wherein the light chain retains at leastone histidine substitution in its expressed light chain variable domainand (b) a non-human, e.g., a mouse, light chain constant region aminoacid sequence, wherein the light chain is associated with a reversechimeric heavy chain comprising (a) a heavy chain variable domainderived from a rearrangement of human V, D, and J segments, wherein theV, D, and J segments are selected from a repertoire of human V, D, and Jsegments present in the animal, and (b) a non-human, e.g., mouse, heavychain constant region amino acid sequence. In one embodiment, therepertoire of human V, D, and J segments comprises at least onefunctional V, at least one functional D, and at least one functional Jsegment, e.g., up to a complete repertoire of functional human V, D, andJ segments. In one embodiment, the heavy and the light chain constantregions are endogenous heavy and light chain constant regions. In oneembodiment, the heavy and light chain variable domains are somaticallymutated domains. In one embodiment, the somatically mutated light chaindomain retains at least one histidine substitution introduced into thegermline sequence. In some embodiments, the somatically mutated lightchain domain retains all or substantially all histidine substitutionsintroduced into the germline sequence. In one embodiment, theantigen-binding protein displays pH-dependent antigen bindingproperties.

In one embodiment, also provided herein is a B cell of the geneticallymodified animal described herein, that comprises in its germline ahistidine-modified human light chain variable region sequence, e.g., ahistidine-modified single rearranged human light chain variable regionsequence, described herein, and expresses an antigen-binding proteindescribed herein. In one embodiment, the antigen-binding protein, e.g.,an antibody, expressed in the B cell retains at least one histidineresidue introduced into the germline, and displays pH-dependentantigen-binding properties. In some embodiments, the antigen-bindingprotein, e.g., an antibody, expressed in the B cell retains all orsubstantially all histidine residues introduced into the germline, anddisplays pH-dependent antigen-binding properties.

In various embodiments, the genetically modified non-human animaldescribed herein comprises a human light chain variable region genesequence, e.g., a single rearranged human light chain variable regiongene sequence (e.g., Vκ1-39Jκ5 or Vκ3-20Jκ1 sequence) that comprises asubstitution of at least one non-histidine codon with a histidine codon(or an addition of a histidine codon into the germline sequence). Theseadditions or substitutions result in a non-human animal that comprises apopulation of B cells enriched for antigen-binding proteins with pHdependent binding properties for their antigens. In one embodiment,antigen-binding proteins, e.g., antibodies, generated in the non-humananimals described herein in response to antigen stimulation display pHdependent antigen binding while exhibiting high affinity for the antigenat neutral pH, e.g., pH between about 7.0 and about 8.0, e.g., pHbetween about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4,e.g., physiological pH. In one embodiment, the affinity of theantigen-binding protein to its antigen, expressed as a dissociationconstant (K_(D)) at a neutral pH is less than 10⁻⁶ M, e.g., less than10⁻⁸ M, e.g., less than 10⁻⁹ M, e.g., less than 10⁻¹⁰ M, e.g., less than10⁻¹¹ M, e.g., less than 10⁻¹² M.

In one embodiment, an antigen-binding protein, e.g., an antibody,generated in the genetically modified non-human animal described herein,exhibits reduced binding to its antigen in acidic pH (e.g., pH of 6.0 orlower, e.g., pH between about 5.0 and about 6.0, pH between about 5.75and about 6.0, e.g., pH of endosomal or lysosomal compartments) ascompared to neutral pH. In one embodiment, the antigen-binding protein,e.g., the antibody, generated in the genetically modified non-humananimal described herein, exhibits no binding to the antigen in acidicpH, while retaining binding to the antigen at neutral pH. In oneembodiment, an antigen-binding protein generated by the geneticallymodified non-human animal described herein, has a decrease indissociative half-life (t_(1/2)) at an acidic pH as compared to thedissociative half-life (t_(1/2)) of the antigen-binding protein at aneutral pH of at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold, at least about 25-fold, or atleast about 30-fold. In one embodiment, an antigen-binding proteinexpressed by the genetically modified non-human animal described hereinhas a t_(1/2) at an acidic pH and 37° C. of about 2 min or less. In oneembodiment, an antigen-binding protein expressed by the geneticallymodified non-human animal described herein has a t_(1/2) at an acidic pHand 37° C. of less than about 1 min. In one embodiment, anantigen-binding protein expressed by the genetically modified non-humananimal described herein has a t_(1/2) at an acidic pH and 25° C. ofabout 2 min or less. In one embodiment, an antigen-binding proteinexpressed by the genetically modified non-human animal described hereinhas a t_(1/2) at an acidic pH and 25° C. of less than about 1 min.

Kinetic parameters, such as equilibrium dissociation constants (K_(D))and dissociative half-lives (t_(1/2)) can be calculated from kineticrate constant as: K_(D) (M)=k_(d)/k_(a); andt_(1/2)(min)=ln2/(60*k_(d)).

In one embodiment, the antigen-binding protein, e.g., an antibody,generated in the genetically modified non-human animals describedherein, exhibits increased binding to FcRn molecule. As described above,FcRn is a receptor present inside the endosomal compartment that iscapable of binding immunoglobulins at an acidic pH and recycling themback to the surface. Screening antibody molecules in the geneticallymodified non-human animals described herein presents a uniqueopportunity to select for antibodies with three beneficial parameters:high affinity for an antigen, pH-dependent antigen binding (with weakerantigen binding at acidic pH) and increased binding to FcRn.

In one embodiment, a genetically modified non-human animal describedherein comprises a population of B cells in response to an antigen thatproduces and is enriched for antigen-binding proteins, e.g., antibodies,that, when reformatted into therapeutics, exhibit increased serum halflife upon administration of a therapeutic dose to a subject over anequivalent B cell population produced in response to the same antigen innon-human animals that do not comprise histidine modification(s) intheir human light chain variable region gene sequences. Thus, in oneembodiment, an antigen-binding protein, e.g., an antibody, produced inresponse to an antigen of interest in a genetically modified non-humananimal described herein, when reformatted into a therapeutic, exhibitsincreased serum half life upon administration of a therapeutic dose to asubject over a serum half life of an antigen-binding protein (whenreformatted into a therapeutic and administered at the same therapeuticdose) that was produced in response to the same antigen in a non-humananimal that does not comprise histidine modification(s) in its humanlight chain variable region gene sequence. In some embodiments, theincrease in serum half life is about 2 fold, e.g., about 5 fold, e.g.,about 10 fold, e.g., about 15 fold, e.g., about 20 fold, or greater.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a non-human as described herein is provided. In a specificembodiment, the cell is an embryonic stem (ES) cell.

In one aspect, a tissue derived from a non-human animal as describedherein is provided. In one embodiment, the tissue is derived fromspleen, lymph node or bone marrow of a non-human animal as describedherein.

In one aspect, a nucleus derived from a non-human animal as describedherein is provided. In one embodiment, the nucleus is from a diploidcell that is not a B cell.

In one aspect, a non-human cell is provided that is isolated from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein. In one embodiment, the cell is an ES cell. In one embodiment,the cell is a lymphocyte. In one embodiment, the lymphocyte is a B cell.In one embodiment, the B cell expresses a chimeric heavy chaincomprising a variable domain derived from a human gene segment; and alight chain derived from a rearranged human Vκ1-39/J sequence with asubstitution of at least one non-histidine codon with histidine codon,rearranged human Vκ3-20/J sequence with a substitution of at least onenon-histidine codon with histidine codon, or a combination thereof andfurther comprising a substitution of at least one amino acid encoded inthe germline for a histidine; wherein the heavy chain variable domain isfused to a non-human or a human heavy chain constant region and thelight chain variable domain is fused to a non-human or a human lightchain constant region.

In one aspect, a hybridoma is provided, wherein the hybridoma is madewith a B cell of a non-human animal as described herein. In a specificembodiment, the B cell is from a mouse as described herein that has beenimmunized with an immunogen comprising an epitope of interest, and the Bcell expresses a binding protein that binds the epitope of interest, thebinding protein has a somatically mutated human variable heavy chaindomain and a mouse C_(H), and has a human variable light chain domainderived from a rearranged human Vκ1-39Jκ5 with a substitution of atleast one non-histidine codon with histidine codon or a rearranged humanVκ3-20Jκ1 with a substitution of at least one non-histidine codon withhistidine codon and a mouse C_(L), wherein the human light chain domaincomprises a substitution of at least one amino acid encoded in thegermline with a histidine.

Also provided is a cell expressing an antigen-binding protein generatedin the non-human animals described herein. In one embodiment, the cellis selected from CHO, COS, 293, HeLa, and a retinal cell expressing aviral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a non-human embryo is provided, wherein the embryocomprises a donor ES cell that is derived from a non-human animal asdescribed herein.

The non-human animals described herein are useful to generate B cellsthat express antibodies having histidines in a CDR3. An animal thatplaces histidines in a CDR3 is useful for making antibodies in general,and in particular useful for developing antibodies that bind a targetwith sufficient affinity at or around a neutral pH, but that either donot bind or that bind weaker to the same target at an acidic pH.

The non-human animal is useful to generate variable regions ofantibodies that can be used to make, e.g., human therapeutic bindingproteins that bind their targets by human immunoglobulin variabledomains that comprise the histidines in a CDR3. The altered binding at alower pH will in some circumstances allow faster turnover because thetherapeutic will bind a target on a cell's surface, be internalized inan endosome, and more readily or more rapidly dissociate from the targetin the endosome, so that the therapeutic can be recycled to bind yetanother molecule of target (e.g., on another cell or the same cell). Insome circumstances, this will result in the ability to dose thetherapeutic at a lower dose, or dose the therapeutic less frequently.This is particularly useful where it is not desirable to dosefrequently, or to administer above a certain dosage, for safety ortoxicity reasons. As a result, the serum half life of the antibodytherapeutic when administered to a subject will be increased.

The non-human animal, e.g., rodent, e.g., mouse or rat, is useful in amethod for increasing the number of B cells in an animal that exhibit anantibody variable region having a CDR3 with one or more histidines init. The non-human animal is useful for generating antibody sequencesthat will exhibit pH-dependent antigen binding. The non-human animal isuseful for generating a greater number of antibody sequences, resultingfrom a single immunization, wherein the antibodies will exhibit apH-dependent antigen binding.

Antigen-Binding Proteins and Methods of Generating the Same

In one aspect, also provided herein are methods for generating humanantigen-binding proteins, e.g., antibodies, which exhibit pH-dependentantigen binding, from the genetically modified non-human animalsdescribed herein with standard methods used in the art.

Several techniques for producing antibodies have been described. Forexample, in various embodiments chimeric antibodies are produced in miceas described herein. Antibodies can be isolated directly from B cells ofan immunized mouse (e.g., see U.S. 2007/0280945A1) and/or the B cells ofthe immunized mouse can be used to make hybridomas (Kohler and Milstein,1975, Nature 256:495-497). DNA encoding the antibodies (human heavyand/or light chains) from non-human animals as described herein isreadily isolated and sequenced using conventional techniques. Hybridomaand/or B cells derived from non-human animals as described herein serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof monoclonal antibodies in the recombinant host cells. The DNA also maybe modified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the non-humansequences. Thus, once nucleic acid sequences of antibodies with desiredcharacteristics, e.g., affinity, epitope, pH-dependent antigen binding,etc., are determined, the non-human constant region gene sequences arereplaced with a desired human constant region sequences to generate afully human antibody containing a non-IgM isotype, for example, IgG1,IgG2, IgG3 or IgG4.

Thus, in one embodiment provided herein is a method of generating anantibody that exhibits pH-dependent antigen binding propertiescomprising generating a non-human animal (e.g., a mouse) as describedherein, immunizing a mouse with an antigen of interest, allowing anon-human animal to mount an immune response to the antigen, andselecting in the non-human animal an antigen-specific antibody thatexhibits pH dependent antigen binding properties, e.g., weaker bindingto the antigen at an acidic than at neutral pH.

Also provided herein are methods of making multi-specific antigenbinding proteins, e.g., bispecific antigen-binding proteins. These aremolecules capable of binding more than one epitope with high affinity.Advantages of the invention include the ability to select suitably highbinding (e.g., affinity matured) heavy chain immunoglobulin chains eachof which will associate with a single light chain. In addition,advantages of the invention include the ability to generate amulti-specific, e.g., a bispecific, antigen-binding protein thatexhibits pH-dependent antigen binding.

Because of the dual nature of bispecific antibodies (i.e., may bespecific for different epitopes of one polypeptide or may containantigen-binding domains specific for more than one target polypeptide,see, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244), they offer many useful advantages fortherapeutic application. For example, the bispecific antibodies can beused for redirected cytotoxicity (e.g., to kill tumor cells), as avaccine adjuvant, for delivering thrombolytic agents to clots, forconverting enzyme activated prodrugs at a target site (e.g., a tumor),for treating infectious diseases, targeting immune complexes to cellsurface receptors, or for delivering immunotoxins to tumor cells.

The bispecific antibodies described herein can also be used in severaltherapeutic and non-therapeutic and/or diagnostic assay methods, suchas, enzyme immunoassays, two-site immunoassays, in vitro or in vivoimmunodiagnosis of various diseases (e.g., cancer), competitive bindingassays, direct and indirect sandwich assays, and immunoprecipitationassays. Other uses for the bispecific antibodies will be apparent tothose skilled in the art.

Several techniques for making bispecific antibody fragments fromrecombinant cell culture have been reported. However, synthesis andexpression of bispecific binding proteins has been problematic, in partdue to issues associated with identifying a suitable light chain thatcan associate and express with two different heavy chains, and in partdue to isolation issues. In various embodiments, compositions andmethods described herein provide the advantage of full length bispecificantibodies that do not require special modification(s) to maintaintraditional immunoglobulin structure by increasing stability/interactionof the components. In various embodiments, such modification(s) hasproven cumbersome and served as an obstacle to development of bispecificantibody technology and their potential use in treating for humandisease. Thus, in various embodiments, through providing a naturalimmunoglobulin structure (i.e., full length) having the added propertyof multiple specificities, full length bispecific antibodies maintaintheir critical effector functions that previous bispecific fragmentslacked, and further provide therapeutics that demonstrate the importantpharmacokinetic parameter of a longer half-life.

Methods and compositions described herein allow for a geneticallymodified mouse to select, through otherwise natural processes, asuitable light chain that can associate and express with more than oneheavy chain, including heavy chains that are somatically mutated (e.g.,affinity matured), wherein the light chain further confers upon theantigen-binding protein its pH-dependent antigen binding property. Humanheavy and light chain variable region sequences from suitable B cells ofimmunized mice as described herein that express affinity maturedantibodies having reverse chimeric heavy chains (i.e., human variableand mouse constant) can be identified and cloned in frame in anexpression vector with a suitable human constant region gene sequence(e.g., a human IgG1). Two such constructs can be prepared, wherein eachconstruct encodes a human heavy chain variable domain that binds adifferent epitope. One of the human light chain variable regions (e.g.,human Vκ1-39Jκ5 or human Vκ3-20Jκ1), comprising a substitution of atleast one non-histidine codon with a histidine codon, can be fused inframe to a suitable human light chain constant region gene (e.g., ahuman κ constant gene). These three fully human heavy and lightconstructs can be placed in a suitable cell for expression. The cellwill express two major species: a homodimeric heavy chain with theidentical light chain, and a heterodimeric heavy chain with theidentical light chain. To allow for a facile separation of these majorspecies, one of the heavy chains is modified to omit a Protein A-bindingdeterminant, resulting in a differential affinity of a homodimericbinding protein from a heterodimeric binding protein. Compositions andmethods that address this issue are described in U.S. Ser. No.12/832,838, filed 25 Jun. 2010, entitled “Readily Isolated BispecificAntibodies with Native Immunoglobulin Format,” published as US2010/0331527A1, hereby incorporated by reference. Once the speciecomprising heterodimeric heavy chain with an identical light chain isselected, this bi-specific antigen binding protein can be screened toconfirm the retention of its pH-dependent antigen binding property.

In one aspect, an epitope-binding protein as described herein isprovided, wherein human light chain and heavy chain variable regionsequences are derived from animals described herein that have beenimmunized with an antigen comprising an epitope of interest.

In one embodiment, an epitope-binding protein is provided that comprisesa first and a second polypeptide, the first polypeptide comprising, fromN-terminal to C-terminal, a first epitope-binding region thatselectively binds a first epitope, followed by a constant region thatcomprises a first C_(H)3 region of a human IgG selected from IgG1, IgG2,IgG4, and a combination thereof; and, a second polypeptide comprising,from N-terminal to C-terminal, a second epitope-binding region thatselectively binds a second epitope, followed by a constant region thatcomprises a second C_(H)3 region of a human IgG selected from IgG1,IgG2, IgG4, and a combination thereof, wherein the second C_(H)3 regioncomprises a modification that reduces or eliminates binding of thesecond C_(H)3 domain to protein A. Various such modifications aredescribed in, e.g., U.S. Application Publication Nos. 2010/0331527 and2011/0195454, incorporated herein by reference.

One method for making an epitope-binding protein that binds more thanone epitope and exhibits pH-dependent epitope binding property is toimmunize a first mouse in accordance with the invention with an antigenthat comprises a first epitope of interest, wherein the mouse comprises(1) an endogenous immunoglobulin light chain variable region locus thatdoes not contain an endogenous mouse light chain variable region genesequence that is capable of rearranging and forming a light chain,wherein at the endogenous mouse immunoglobulin light chain variableregion locus is a single rearranged human light chain variable regionoperably linked to the mouse endogenous light chain constant regiongene, and the rearranged human light chain variable region is selectedfrom a human Vκ1-39Jκ5 and a human Vκ3-20Jκ1 comprising a substitutionof at least one non-histidine codon with a histidine condon, and (2) theendogenous mouse V_(H) gene segments have been replaced in whole or inpart with human V_(H) gene segments, such that immunoglobulin heavychains made by the mouse are solely or substantially heavy chains thatcomprise human variable domains and mouse constant domains. Whenimmunized, such a mouse will make a reverse chimeric antibody,comprising only one of two human light chain variable domains (e.g., oneof human Vκ1-39Jκ5 or human Vκ3-20Jκ1, e.g., comprising a substitutionof at least one amino acid with a histidine). Commonly, at least some ofthe substituted histidine residues introduced into the germline sequencewill be retained in the reverse chimeric antibody. Once a B cell isidentified that encodes a heavy chain variable domain that binds theepitope of interest and expresses an antibody that exhibits pH-dependentantigen binding properties, the nucleotide sequence of the heavy chainvariable region (and, optionally, the light chain variable region) canbe retrieved (e.g., by PCR) and cloned into an expression construct inframe with a suitable human immunoglobulin heavy chain constant regionsequence. This process can be repeated to identify a second heavy chainvariable domain that binds a second epitope, and a second heavy chainvariable region gene sequence can be retrieved and cloned into anexpression vector in frame to a second suitable human immunoglobulinheavy chain constant region sequence. The first and the secondimmunoglobulin constant domains encoded by the constant region genesequence can be the same or different isotype, and one of theimmunoglobulin constant domains (but not the other) can be modified asdescribed herein or in US 2010/0331527A1, and epitope-binding proteincan be expressed in a suitable cell and isolated based on itsdifferential affinity for Protein A as compared to a homodimericepitope-binding protein, e.g., as described in US 2010/0331527A1.

Thus, in various embodiments, following isolation of the DNA andselection of the first and second nucleic acid sequences that encode thefirst and second human heavy chain variable domains having the desiredspecificities/affinities, and a third nucleic acid sequence that encodesa human light chain domain (a germline rearranged sequence or a lightchain sequence isolated from a non-human animal as described herein) andcomprises a substitution of at least one non-histidine codon with ahistidine codon, the three nucleic acids sequences encoding themolecules are expressed to form the bispecific antibody usingrecombinant techniques which are widely available in the art. Often, theexpression system of choice will involve a mammalian cell expressionvector and host so that the bispecific antibody is appropriatelyglycosylated (e.g., in the case of bispecific antibodies comprisingantibody domains which are glycosylated). However, the molecules canalso be produced in the prokaryotic expression systems. Normally, thehost cell will be transformed with DNA encoding both the first humanheavy chain variable domain, the second human heavy chain variabledomain, the human light chain domain on a single vector or independentvectors. However, it is possible to express the first human heavy chainvariable domain, second human heavy chain variable domain, and humanlight chain domain (the bispecific antibody components) in independentexpression systems and couple the expressed polypeptides in vitro. Invarious embodiments, the human light chain domain is derived from agermline sequence but for the substitution of at least one non-histidinecodon with a histidine codon, e.g., in a CDR codon. In variousembodiments, the human light chain domain comprises no more than one, nomore than two, no more than three, no more than four, or no more thanfive somatic hypermutations within the light chain variable sequence ofthe light chain domain. In some embodiments, the somatic hypermutationsdo not alter the presence of at least one histidine residue introducedinto the germline sequence of the light chain variable region.

In various embodiments, the nucleic acid(s) (e.g., cDNA or genomic DNA)encoding the two heavy chains and single human light chain with asubstitution of at least one non-histidine with a histidine is insertedinto a replicable vector for further cloning (amplification of the DNA)and/or for expression. Many vectors are available, and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Each component may be selected individually or based on a host cellchoice or other criteria determined experimentally. Several examples ofeach component are known in the art.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid sequences that encode each or all the components of the bispecificantibody. A large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto bispecific antibody-encoding DNA by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) may also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the bispecific antibody components.Suitable expression vectors for various embodiments include those thatprovide for the transient expression in mammalian cells of DNA encodingthe bispecific antibody. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of bispecific antibodieshaving desired binding specificities/affinities or the desired gelmigration characteristics relative to the parental antibodies havinghomodimers of the first or second human heavy chain variable domains.

In various embodiments, once the DNA encoding the components of thebispecific antibody are assembled into the desired vector(s) asdescribed above, they are introduced into a suitable host cell forexpression and recovery. Transfecting host cells can be accomplishedusing standard techniques known in the art appropriate to the host cellselected (e.g., electroporation, nuclear microinjection, bacterialprotoplast fusion with intact cells, or polycations, e.g., polybrene,polyornithine, etc.).

A host cell is chosen, in various embodiments, that best suits theexpression vector containing the components and allows for the mostefficient and favorable production of the bispecific antibody species.Exemplary host cells for expression include those of prokaryotes andeukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In various embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In various embodiments,the cell is eukaryotic cell selected from CHO (e.g., CHO K1, DXB-11 CHO,Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, W138, MRC 5,Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal),CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertolicell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cellline derived from an aforementioned cell. In various embodiments, thecell comprises one or more viral genes, e.g. a retinal cell thatexpresses a viral gene (e.g., a PER.C6™ cell).

Mammalian host cells used to produce the bispecific antibody may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. Media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other supplements may also be includedat appropriate concentrations as known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are, invarious embodiments, those previously used with the host cell selectedfor expression, and will be apparent to those skilled in the art.

The bispecific antibody may be recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysate when directly produced without a secretory signal. If thebispecific antibody is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g., Triton-X 100).

Following isolation, a bispecific antibody comprising a two human heavychains and a single human light chain derived from a rearranged humanlight chain variable region gene sequence selected from Vκ1-39Jκ5 andVκ3-20Jκ1 sequences that comprise a substitution of at least onenon-histidine codon with a histidine codon, is screened for its abilityto exhibit pH dependent binding to one, preferably both of its antigens.The ability of bispecific antibodies to bind its antigens differently atneutral and acidic pH's (e.g., their ability to demonstrate decreasedt_(1/2) at acidic pH compared to neutral pH) can be determined by avariety of techniques available in the art and described in thefollowing examples, e.g., BIACORE™ assay.

Additional Methods for Generating Antigen-Binding Proteins withpH-Dependent Antigen Binding

Various methods of generating antigen-binding proteins with pH-dependentantigen binding properties in genetically modified non-human animalsdescribed herein are provided. Also provided are methods of generatingantigen binding proteins with pH-dependent antigen binding properties invitro. Such methods may involve generating various components of theantigen-binding proteins in vivo in genetically modified non-humananimals, and then modifying them and reassembling them in vitro outsidean organism as protein complexes expressed in mammalian cell culture.

In one embodiment, the method of generating antigen-binding proteinswith pH-dependent antigen binding properties utilizes an antigen-bindingprotein sequence, e.g., an antibody sequence, that is generated in amouse comprising a limited repertoire of light chain variable region Vand J segments, e.g., human light chain variable region V and Jsegments, “universal light chain” or “common light chain” mouse (“ULC”mouse), such as the mouse described in U.S. Application Publication Nos.2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492, allincorporated herein by reference. In one embodiment, the method ofgenerating antigen-binding proteins with pH-dependent antigen bindingproperties utilizes an antigen binding protein sequence that isgenerated in a mouse comprising a single rearranged human light chainvariable region gene sequence. In one embodiment, the method utilizes anantigen binding protein generated in a mouse comprising a singlerearranged human light chain variable region gene sequence selected fromhuman Vκ1-39Jκ5 and human Vκ3-20Jκ1.

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH dependent antigen binding propertiescomprises selecting a first antibody that binds to an antigen ofinterest (e.g., binds to an antigen of interest with a desiredaffinity), modifying an immunoglobulin light chain nucleotide sequenceof the first antibody to comprise a substitution of at least onenon-histidine codon with a histidine codon, expressing an immunoglobulinheavy chain of the first antibody and the modified immunoglobulin lightchain in a cell, and selecting a second antibody expressed in the cellthat retains binding to the antigen of interest (e.g., retains desiredaffinity for the antigen of interest) at neutral pH and displays reducedbinding to the antigen of interest at an acidic pH.

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH dependent antigen binding propertiescomprises selecting an immunoglobulin heavy chain from an antibody(e.g., obtained from a non-human animal, e.g., a mouse, e.g., a ULCmouse) that comprises an immunoglobulin light chain having a singlerearranged human immunoglobulin light chain variable region sequencewherein the antibody binds to an antigen of interest (e.g., binds to anantigen of interest with a desired affinity); modifying the nucleic acidsequence of the immunoglobulin light chain such that the singlerearranged human immunoglobulin light chain variable region sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon; expressing the selected immunoglobulin heavy chain andthe immunoglobulin light chain comprising the substitution of at leastone amino acid with a histidine in its variable domain; and selecting anantibody that retains binding to the antigen of interest at a neutral pH(e.g., retains desired affinity to the antigen of interest) whiledisplaying reduced binding to the antigen of interest at an acidic pH.In various embodiments, the immunoglobulin heavy chain is derived from arearrangement of human heavy chain variable gene segments (human V, D,and J segments).

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH-dependent antigen binding propertiescomprises (1) immunizing a non-human animal, e.g., a mouse, comprising asingle rearranged human light chain variable region gene sequence and arepertoire of unrearranged human heavy chain variable gene segments (V,D, and J segments) with an antigen of interest and allowing a mouse tomount an immune response to said antigen, (2) selecting in the non-humananimal, e.g., in the mouse, an antibody that binds to the antigen ofinterest with a desired affinity, (3) isolating from the non-humananimal, e.g., from the mouse, a nucleotide sequence of an immunoglobulinheavy chain of the antibody that binds to the antigen of interest with adesired affinity, (4) determining the nucleotide sequence of said heavychain, (5) modifying a nucleotide sequence of an immunoglobulin lightchain containing the single rearranged human immunoglobulin light chainvariable region to comprise a substitution of at least one non-histidinecodon with a histidine codon, (6) expressing the immunoglobulin heavychain of the antibody that binds to the antigen of interest with desiredaffinity and the immunoglobulin light chain comprising the histidinemodification in a cell, and (7) determining whether the antibodyexpressed in the cell retains binding to the antigen at a neutral pHwhile displaying reduced binding at an acidic pH. In one embodiment, theantibody expressed in the cell exhibits desired affinity to the antigenat neutral pH. In various embodiments, the immunoglobulin heavy chain isderived from a rearrangement of human heavy chain variable gene segments(human V, D, and J segments).

In one embodiment, the mouse comprising a single rearranged human lightchain variable region gene sequence is a universal light chain or commonlight chain “ULC” mouse described in, e.g., U.S. Application PublicationNos. 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492. In oneembodiment, the single rearranged human light chain variable region genesequence is selected from human Vκ1-39Jκ5 and human Vκ3-20Jκ1 sequence.

In one embodiment, the antigen of interest is selected from a solubleantigen, a cell surface antigen (e.g., a tumor antigen) and a cellsurface receptor. In a specific embodiment, the cell surface receptor isan immunoglobulin receptor. In a specific embodiment, the immunoglobulinreceptor is an Fc receptor.

In one embodiment, the desired affinity of an antibody for an antigenexpressed as a dissociation constant (K_(D)) at a neutral pH is lessthan 10⁻⁶ M, e.g., less than 10⁻⁸ M, e.g., less than 10⁻⁹ M, e.g., lessthan 10⁻¹⁰ M, e.g., less than 10⁻¹¹ M, e.g., less than 10⁻¹² M.

As explained above, the ULC mice, in one embodiment, comprise a singlerearranged human immunoglobulin light chain variable gene sequence, andexpress antibodies in response to the antigen where the affinity ofantibodies to the antigen is primarily mediated through the heavy chainsof their antibodies. These mice comprise a repertoire of human heavychain variable (V, D, and J) segments, that rearrange to encode a humanheavy chain variable domain of an antibody that also comprises the lightchain derived from the single rearranged human light chain variablesequence. In one embodiment, upon antigen exposure, these mice utilizethe diverse repertoire of human heavy chain variable (V, D, and J)segments to generate an antibody with affinity to and specificity forthe antigen. Thus, upon exposure to the antigen, the nucleotide sequenceof an immunoglobulin heavy chain of the antibody generated in the ULCmice may be isolated and utilized to generate a desired binding proteinalso comprising an immunoglobulin light chain derived from the singlerearranged human immunoglobulin light chain variable region sequence(e.g., the single rearranged human immunoglobulin light chain variableregion sequence with a substitution of at least one non-histidine codonwith a histidine codon).

In one embodiment of the ULC mice, 90-100% of unrearranged non-humanV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially all(e.g., 90-100%) of the endogenous non-human V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In oneembodiment, the replacement is with at least 19, at least 39, or atleast 80 or 81 unrearranged human V_(H) gene segments. In oneembodiment, the replacement is with at least 12 functional unrearrangedhuman V_(H) gene segments, at least 25 functional unrearranged humanV_(H) gene segments, or at least 43 functional unrearranged human V_(H)gene segments. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with at least oneunrearranged human D_(H) segment and at least one unrearranged humanJ_(H) segment. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with allunrearranged human D_(H) segments and all unrearranged human J_(H)segments. Thus, the ULC mouse utilizes a diverse repertoire of humanvariable region gene segments (V, D, and J segments) to generate anantibody in response to the antigen of interest.

Once the heavy chain of the antibody that binds to the antigen ofinterest with the desired affinity is determined, the nucleotidesequence of the heavy chain is isolated and sequenced. The sequence iscloned into a vector for expression in suitable host cells, e.g.,eukaryotic cells, e.g., CHO cells. In one embodiment, the sequence of ahuman heavy chain constant region is cloned downstream of the humanheavy chain variable region sequence isolated from the mouse (e.g., fromthe ULC mouse).

In one embodiment, the method of generating an antigen-binding proteinwith pH-dependent antigen-binding properties comprises modifying anucleotide sequence of the immunoglobulin light chain, particularly thesequence of the single rearranged human immunoglobulin light chainvariable region, to comprise a substitution of at least onenon-histidine codon with a histidine codon. Various techniques formodifying a nucleotide sequence are known in the art, e.g., sitedirected mutagenesis. In addition, a nucleotide sequence comprising thedesired histidine substitution may be synthesized de novo.

In one embodiment, the substitution of at least one non-histidine codonwith a histidine codon comprises a substitution resulting in expressionof one, two, three, four, or more histidine residues. In one embodiment,the substitution(s) results in expression of three or four histidineresidues. In one embodiment, the substitution(s) is in theimmunoglobulin light chain variable region. In one embodiment, thesubstitution(s) is in the CDR codon, e.g., CDR1, CDR3, and/or CDR3codon. In one embodiment, the substitution(s) is in the CDR3 codon.

In one embodiment, wherein the immunoglobulin light chain nucleic acidsequence comprises Vκ1-39Jκ5 gene sequence, and the substitution(s) isin the CDR3 codon, the substitution results in expression of a histidineat position selected from 105, 106, 108, 111, and combinations thereof.In one embodiment, the substitutions result in expression of histidinesat positions 105, 106, 108, and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and106. In one embodiment, the substitutions result in expression ofhistidines at positions 105 and 108. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and111. In one embodiment, the substitutions result in expression ofhistidines at positions 106 and 108. In one embodiment, thesubstitutions result in expression of histidines at positions 106 and111. In one embodiment, the substitutions result in expression ofhistidines at positions 108 and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 106,and 108. In one embodiment, the substitutions result in expression ofhistidines at positions 105, 106, and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 108,and 111. In one embodiment, the substitutions result in expression ofhistidines at positions 106, 108, and 111. Amino acid and nucleic acidsequences of Vκ1-39Jκ5 CDR3 regions comprising various histidinesubstitutions are depicted in FIG. 2 and included in the sequencelisting.

In one embodiment, wherein the immunoglobulin light chain nucleic acidsequence comprises Vκ3-20Jκ1 gene sequence, and the substitution(s) isin the CDR3 codon, the substitution results in expression of a histidineat position selected from 105, 106, 107, 109, and combinations thereof.In one embodiment, the substitutions result in expression of histidinesat positions 105, 106, 107, and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and106. In one embodiment, the substitutions result in expression ofhistidines at positions 105 and 107. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and109. In one embodiment, the substitutions result in expression ofhistidines at positions 106 and 107. In one embodiment, thesubstitutions result in expression of histidines at positions 106 and109. In one embodiment, the substitutions result in expression ofhistidines at positions 107 and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 106,and 107. In one embodiment, the substitutions result in expression ofhistidines at positions 105, 106, and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 107,and 109. In one embodiment, the substitutions result in expression ofhistidines at positions 106, 107, and 109. Selected amino acid andnucleic acid sequences of Vκ3-20Jκ1 CDR3 regions comprising varioushistidine substitutions are depicted in FIG. 12 and included in thesequence listing.

Once the sequence of immunoglobulin light chain, e.g., humanimmunoglobulin light chain variable domain, is modified to includehistidine residues at desired positions, the nucleotide sequence of thelight chain is cloned into a vector for expression in suitable hostcells, e.g., eukaryotic cells, e.g., CHO cells. In one embodiment, thesequence of a human light chain constant region is cloned downstream ofthe modified nucleotide sequence of human variable region.

In one embodiment, vectors comprising nucleotide sequence encodingmodified human immunoglobulin light chain and selected humanimmunoglobulin heavy chain are co-expressed in a suitable host cell,e.g., eukaryotic host cell, e.g., CHO cell, to generate anantigen-binding protein. Various host cells that can be used forexpression are known in the art and are mentioned throughout thisspecification.

An antigen-binding protein, e.g., an antibody, generated in the hostcell may be secreted into cell supernatant, which is screened for properexpression and affinity for the original antigen at neutral pH. Theantigen-binding protein may also be recovered from cell lysate, or, ifmembrane bound, released from the membrane using a suitable detergent(e.g., Triton-X). The antigen-binding protein with desiredcharacteristics may be purified.

In one embodiment, the antigen-binding protein comprising histidinemodification(s) retains the affinity to the antigen that is comparableto the affinity to the antigen of the same (original) antigen-bindingprotein that does not comprise histidine modification(s). In oneembodiment, the affinity of the histidine-modified antigen-bindingprotein for the antigen of interest expressed as a dissociation constant(K_(D)) at a neutral pH is less than 10⁻⁶ M, e.g., less than 10⁻⁸ M,e.g., less than 10⁻⁹ M, e.g., less than 10⁻¹⁰ M, e.g., less than 10⁻¹¹M, e.g., less than 10⁻¹² M.

In one embodiment, the antigen-binding protein, e.g., an antibody,comprising histidine modifications described herein exhibits pHdependent antigen binding properties. In one embodiment, theantigen-binding protein comprising histidine modifications possessesenhanced pH dependent properties over an equivalent antigen-bindingprotein without the histidine modifications (antigen-binding protein ofthe same amino acid sequence but for the histidine modifications). Inone embodiment, the antigen-binding protein described herein retainsbinding to the antigen at neutral pH (e.g., retains desired affinity forthe antigen at neutral pH) while displaying reduced binding at an acidicpH. In one embodiment, the antigen-binding protein, e.g., the antibody,described herein, exhibits no binding to the antigen in acidic pH, whileretaining binding to the antigen at neutral pH. In one embodiment, anantigen-binding protein described herein, has a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to the dissociativehalf-life (t_(1/2)) of the antigen-binding protein at a neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.In one embodiment, an antigen-binding protein described herein has at_(1/2) at an acidic pH and 37° C. of about 2 min or less. In oneembodiment, an antigen-binding protein described herein has a t_(1/2) atan acidic pH and 37° C. of less than about 1 min. In one embodiment, anantigen-binding protein described herein has a t_(1/2) at an acidic pHand 25° C. of about 2 min or less. In one embodiment, an antigen-bindingprotein described herein has a t_(1/2) at an acidic pH and 25° C. ofless than about 1 min.

In one embodiment, the antigen-binding protein e.g., the antibody,comprising histidine modifications described herein, exhibits increasedserum half life upon administration of a therapeutic dose to a subjectas compared to a serum half life upon administration of an equivalenttherapeutic dose of antigen-binding protein that does not comprisehistidine modifications (e.g., the original antigen-binding protein thatdoes not comprise histidine modifications). In some embodiments, theincrease in serum half life upon administration of a dose of theantigen-binding protein comprising histidine modifications describedherein over a serum half life upon administration of the same dose ofthe antigen-binding protein not comprising histidine modifications isabout 2 fold, e.g., about 5 fold, e.g., about 10 fold, e.g., about 15fold, e.g., about 20 fold, or greater. In one embodiment, serumhalf-life is at least about 1 day, e.g., at least about 2 days, e.g., atleast about 7 days, e.g., at least about 14 days, e.g., at least about30 days, e.g., at least about 60 days.

In addition to the in vitro methods for generating antigen-bindingproteins with pH-dependent antigen binding properties described above,also provided herein are antigen-binding proteins, e.g., antibodies,generated by said method. In addition, said method may be utilized togenerate multi-specific, e.g., bispecific, antigen-binding proteins, byselecting two different human immunoglobulin heavy chains that bind to acommon (universal) light chain in a mouse, determining nucleotidesequences of the heavy chains, modifying universal light chain tocomprise histidine substitutions as described above, and co-expressingtwo human heavy chains with a single histidine-modified universal lightchain in a host cell. Various steps for generating an antigen-bindingprotein described above may be applicable to the method of generating abispecific antigen-binding protein. Bispecific antigen binding protein,confirmed to possess desired affinity for the antigen(s) andpH-dependent antigen binding properties may be purified. Thus,bispecific antibodies comprising two human heavy chains and a singlehuman light chain comprising a human light chain variable domainsequence encoded by a human variable region gene, e.g., Vκ1-39Jκ5 orVκ3-20Jκ1 variable region gene comprising a substitution of at least onenon-histidine codon with a histidine codon, is provided.

Also provided are constructs utilized in making an antigen-bindingprotein comprising human immunoglobulin heavy chain and humanimmunoglobulin light chain comprising histidine substitutions. Hostcells expressing antigen-binding proteins, e.g., antibodies, describedherein are also provided.

Examples

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for. TheExamples do not include detailed descriptions of conventional methodsthat would be well known to those of ordinary skill in the art(molecular cloning techniques, etc.). Unless indicated otherwise, partsare parts by weight, molecular weight is average molecular weight,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1 Identification of Histidine Residues in Antigen-Specific HumanLight Chains

Generation of a common light chain mouse (e.g., Vκ1-39 or Vκ3-20 commonlight chain mouse) and antigen-specific antibodies in those mice isdescribed in U.S. patent application Ser. Nos. 13/022,759, 13/093,156,and 13/412,936 (Publication Nos. 2011/0195454, 2012/0021409, and2012/0192300, respectively), incorporated by reference herein in theirentireties. Briefly, rearranged human germline light chain targetingvector was made using VELOCIGENE® technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela et al. (2003) High-throughput engineering ofthe mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6): 652-659) to modify mouse genomic BacterialArtificial Chromosome (BAC) clones, and genomic constructs wereengineered to contain a single rearranged human germline light chainregion and inserted into an endogenous κ light chain locus that waspreviously modified to delete the endogenous κ variable and joining genesegments. Targeted BAC DNA was then used to electroporate mouse ES cellsto create modified ES cells for generating chimeric mice that express arearranged human germline Vκ1-39Jκ5 or Vκ3-20Jκ1 region. Targeted EScells were used as donor ES cells and introduced into an 8-cell stagemouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No.7,294,754 and Poueymirou et al. (2007) F0 generation mice that areessentially fully derived from the donor gene-targeted ES cells allowingimmediate phenotypic analyses Nature Biotech. 25(1): 91-99). VELOCIMICE®independently bearing an engineered human germline Vκ1-39Jκ5 orVκ3-20Jκ1 light chain region were identified by genotyping using amodification of allele assay (Valenzuela et al., supra) that detects thepresence of the unique rearranged human germline light chain region.

Mice bearing an engineered human germline light chain locus (ULC mice)were bred with mice that contain a replacement of the endogenous mouseheavy chain variable gene locus with the human heavy chain variable genelocus (see U.S. Pat. No. 6,596,541; the VELOCIMMUNE® mouse, RegeneronPharmaceuticals, Inc.).

VELOCIMMUNE® mouse containing a single rearranged human germline lightchain region is challenged with an antigen of interest and antibodiescomprising a universal light chain (e.g., Vκ1-39Jκ5) are isolated andsequenced. Amino acid sequences of selected light chains (A-K,corresponding to SEQ ID NOs: 82-92, respectively) from antigen-specifichuman antibodies generated in a common Vκ1-39Jκ5 light chain mouse werealigned. Histidine mutations in the CDRs of human Vκ1-39-derived lightchains for a selected number of antigen-specific human antibodies wereidentified (FIG. 1). The partial amino acid sequence of germlineVκ1-39Jκ5 variable domain is shown above the alignments and set forth inSEQ ID NO:1, the complete variable domain amino acid sequence is setforth in SEQ ID NO:80.

Example 2 Engineering and Characterization of Histidine-SubstitutedHuman Universal Light Chain Antibodies Example 2.1 Engineering ofHistidine Residues into a Germline Human Rearranged Light Chain

Histidine residues were engineered into a rearranged human Vκ1-39Jκ5light chain using site directed mutagenesis primers specificallydesigned to introduce engineered histidine residues at Q105, Q106, Y108,and P111 positions of the human Vκ1-39Jκ5 light chain. Site directedmutagenesis was performed using molecular techniques known in the art(e.g., QuikChange II XL Site Directed Mutagenesis Kit, AgilentTechnologies). Locations of the engineered residues in the CDR3 areshown in FIG. 2, the nucleic acid sequences of histidine-substitutedCDR3's depicted in FIG. 2 are set forth in SEQ ID NOs: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 (corresponding amino acidsequences are set forth in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, and 33). The nucleic acid and amino acid sequencesof germline rearranged Vκ1-39Jκ5 CDR3 are set forth in SEQ ID NOs: 2 and3, respectively.

Example 2.2 Construction and Expression of Histidine Engineered LightChains

Human Vκ1-39-derived light chains containing germline engineeredhistidine residues made according to Example 2 were constructed andpaired with various human heavy chains (labeled 1-5), specific for ahuman cell surface receptor, to analyze expression in CHO cells. Thefive human heavy chains specific for a human cell surface receptor thatwere paired with histidine-substituted Vκ1-39-derived light chains wereobtained from mice that have a single rearranged human light chain (ahuman Vκ1-39/Jκ5 rearranged light chain; see US2011/0195454A1).

Enzyme-Linked Immunosorbent Assay (ELISA):

Antibody secretion from CHO cells was detected using an Fc ELISA, forlight chains with indicated histidine modifications with five differentheavy chains. The light and heavy chain sequences (but for themodifications) were generated in mice that have a single rearrangedhuman light chain (e.g., a human Vκ1-39/Jκ5 rearranged light chain; seeUS2011/0195454A1). Capture antibody was goat anti-human IgG anddetection antibody was goat anti-human (Fc gamma-specific)-HRP. Theresults are shown in FIG. 3. ULC+heavy: specific heavy chain andunmodified human Vκ1-39-derived light chain. As shown in FIG. 3,expression was detected in about all mutants.

Protein Immunoblot.

Expression in supernatants of CHO cells of paired antigen-specific heavychains with histidine engineered light chains was further analyzed bywestern blot. Samples were run on a 4-12% tris-glycine gel. Resultsusing a selected heavy chain (heavy chain 3) are shown in FIG. 4. ULCrefers to a rearranged human Vκ1-39-derived light chain (as describedabove).

Example 2.3 Determination of Binding Affinity of Histidine EngineeredLight Chains

Equilibrium dissociation constants (K_(D)), dissociative half-lives(t_(1/2)), and other kinetic parameters for selected antibodysupernatants were determined by SPR (Surface Plasmon Resonance) using aBIACORE™ T200 instrument (GE Healthcare). Kinetics were measured at pH7.4 and at pH 5.75. Results are shown in FIGS. 5A-5E.

Numerical values for the kinetic binding properties (e.g., k_(a), k_(d),K_(D), t_(1/2), etc.) of antibodies binding to immunogen at neutral pH(pH 7.4) and at acidic pH (pH 5.75) were obtained using a real-timesurface plasmon resonance biosensor (Biacore T200.) A Biacore CM5 sensorchip was derivatized with a mouse anti-human Fc antibody to captureantibodies from the supernatant. A single concentration (50 nM) ofimmunogen was then injected over the antibody-captured surface at a flowrate of 30 μl/min. Antibody-antigen association was monitored for 2.5minutes and then the dissociation of antigen from the captured antibodywas monitored for 8 minutes. Kinetic association (ka) and dissociation(kd) rate constants were determined by processing and fitting the datato a 1:1 binding with a mass transport model using Biacore T200Evaluation software version 1.0. Equilibrium dissociation constants(K_(D)) and dissociative half-lives (t_(1/2)) were calculated from thekinetic rate constants as: K_(D) (M)=k_(d)/k_(a); andt_(1/2)(min)=(ln2/(60*k_(d)).

As shown in FIG. 5, in a binding assay of antibody to a cell surfacereceptor, two out of five antibodies with histidine-modified commonlight chains (histidine modified CDR3's of Vκ1-39/Jκ5 light chains) thatwere paired with the antigen-specific human heavy chains, exhibitedbinding to the antigen (e.g., to a cell surface receptor) with differentaffinities at pH 7.4 and pH 5.75. Antibodies with histidinemodifications that retain binding at pH 7.4, but that exhibit a lowbinding or no detectable binding at pH 5.75, are desirable. Antibodieswith histidine modification that exhibit reduced t_(1/2) at pH 5.75 ascompared to pH 7.4 are desirable.

Antigen binding data for three antibodies comprising histidine-modifiedcommon light chains and three antigen-specific heavy chains (labeled 2,3, and 6) at different pHs is summarized further in FIG. 6. Theseantibodies exhibited significant drop in antigen binding at pH 5.75 incomparison to pH 7.4, as demonstrated, e.g., by reduction in t_(1/2) orno binding detected at pH 5.75.

Example 3 Engineering and Characterization of Genetically Modified MouseComprising a Histidine-Substituted Vκ1-39Jκ5 Universal Light ChainExample 3.1 Constructing of Targeting Vector for Engineering HistidineResidues in a Rearranged Human Light Chain Variable Region

A genetically modified mouse containing a rearranged human light chaingene having histidine residues engineered into a CDR region of the humanlight chain is made using targeting vectors made by standard molecularcloning techniques known in the art.

Briefly, various rearranged human germline light chain targeting vectorsare made using VELOCIGENE® technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela et al. (2003) High-throughput engineering ofthe mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6):652-659) to modify mouse genomic BacterialArtificial Chromosome (BAC) DNA to contain a single rearranged humangermline light chain region and inserted into an endogenous κ lightchain locus that was previously modified to delete the endogenous κvariable and joining gene segments. The rearranged human germline lightchain region is modified at one or more nucleotide positions within thesequence of the light chain to encode histidine residues that are notnormally present at the respective locations of the germline sequence.The targeting vectors are electroporated into mouse embryonic stem (ES)cells and confirmed using a quantitative PCR assay (e.g., TAQMAN™).

Specifically, a strategy for constructing these targeting vectors isshown in FIGS. 8A-8F. A plasmid used for generating a targeting vectorfor common (universal) light chain mouse (“ULC mouse,” described in,e.g., US2011/0195454A1), containing pBS+FRT-Ub-Hyg-FRT+mouse Vκ3-7leader+human Vκ1-39Jκ5 was modified by site directed mutagenesis(QuickChange II XL Kit) to replace Q105, Q106, Y108 and P111 or Q106,Y108 and P111 with histidine residues in the CDR3 region usingsite-directed mutagenesis primers shown in FIG. 7 (See FIG. 8A for thisengineering step). Resultant vectors (H105/106/108/111 and H106/108/111)were modified further and ligated into a vector comprising mouse Igκconstant region, mouse enhancers, a mouse 3′ homology arm and a SPECcassette (FIG. 8B). Further modification involved ligation into a vectorcarrying 5′ mouse arm and comprising Frt-Ub-NEO-Frt cassette (FIG. 8B).Resultant targeting vectors were electroporated into ES cells comprisingdeletion of the mouse Igκ variable locus (comprising κ variable andjoining gene segments) (FIGS. 8C-8F).

Positive ES cell clones were confirmed by using a modification of alleleassay (Valenzuela et al.) using probes specific for the engineeredVκ1-39Jκ5 light chain region inserted into the endogenous κ light chainlocus. Primers and probes used in the assay are shown in Table 1 belowand set forth in the Sequence Listing; the locations of the probes aredepicted in FIGS. 8C-8F.

TABLE 1 Primers and Probes Used for ES Cell Screening Probe Probe 5′ 3′Name Assay Sequence Primer Primer Neo GOA TGGGCACA GGTGGAGA GAACACGGACAGACAA GGCTATTC CGGCATCA TCGGCTG GGC G (SEQ ID (SEQ ID (SEQ ID NO: 38)NO: 39) NO: 40) ULC-m1 GOA CCATTATG AGGTGAGG TGACAAAT ATGCTCCA GTACAGATGCCCTAAT TGCCTCTC AAGTGTTA TATAGTGA TGTTC TGAG TCA (SEQ ID (SEQ ID(SEQ ID NO: 41) NO: 42) NO: 43) 1633h2 GOA ATCAGCAG GGGCAAGT TGCAAACT(Vκ1- AAACCAGG CAGAGCAT GGATGCAG 39Jκ5- GAAAGCCC TAGCA CATAG spe- CT(SEQ ID (SEQ ID cific) (SEQ ID NO: 45) NO: 46) NO: 44) mlgKd2 Reten-GGCCACAT GCAAACAA CTGTTCCT tion TCCATGGG AAACCACT CTAAAACT TTC GGCCGGACTCCA (SEQ ID (SEQ ID CAGTAAAT NO: 47) NO: 48) GGAAA (SEQ ID NO: 49)mlgKp15 Reten- GGGCACTG CACAGCTT AGAAGAAG tion GATACGAT GTGCAGCCCCTGTACT GTATGG TCC ACAGCATC (SEQ ID (SEQ ID CGTTTTAC NO: 50) NO: 51)AGTCA (SEQ ID NO: 52)

The NEO selection cassette introduced by the targeting constructs wasdeleted by transfecting ES cells with a plasmid that expresses FLP(FIGS. 8C and 8E). Optionally, the neomycin cassette may be removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99. VELOCIMICE® independently bearing an engineeredhuman light chain gene that contains histidine residues mutated into oneor more positions along the sequence were made from the targeted EScells described above.

Pups were genotyped and pups heterozygous for the engineeredhistidine-modified human light chain were selected for characterizingexpression of the light chain and binding capabilities of the expressedantibodies. Primers and probes for genotyping of mice specificallycomprising a universal light chain gene with either three (H106/108/111;“1930”) or four (H105/105/108/111; “1927”) histidine modifications arelisted in Table 2 below and set forth in the Sequence Listing. Micecontaining histidine modification in their universal light chains arereferred herein as “HULC” mice (histidine universal light chain mice).

TABLE 2 Primers and Probes Used for Genotyping Probe Probe 5′ 3′ NameAssay Sequence Primer Primer 1927jxn3 GOA ACCATAGT AGCAGTCT CCCTTGGC1927 CACAGTAC GCAACCTG CGAAGGTG (4 His) CCA AAGATTT AT mouse- (SEQ ID(SEQ ID (SEQ ID specific NO: 53) NO: 54) NO: 55) 1930jxn3 GOA ATAGTCACAGTCTGCA CCCTTGGC 1930 AGTACCCA ACCTGAAG CGAAGGTG (3 His) TCC ATTTTGC ATmouse- (SEQ ID (SEQ ID (SEQ ID specific NO: 56) NO: 57) NO: 58)

Example 3.2 Analysis of Immune Response to Antigen in Mice withHistidine-Substituted Universal Light Chains

Cell surface receptor (“Antigen A”) was used as the immunogen toimmunize mice that were either heterozygous for expression of apre-arranged human kappa light chain utilizing Vk1-39 and Jk5 that has 4histidine substitutions in CDR3 (hereinafter “HULC 1927”) orheterozygous for expression of a pre-arranged human kappa light chainutilizing Vk1-39 and Jk5 that has 3 histidine substitutions in CDR3(hereinafter “HULC1930”), or homozygous WT mice. Pre-immune serum wascollected from the mice prior to the initiation of immunization. Theimmunogen was administered at 2.35 μg of protein for the initial primingimmunization mixed with 10 μg of CpG oligonucleotide as an adjuvant(Invivogen) in a volume of 25 μl via footpad (f.p.). Subsequently, micewere boosted via the same route with 2.35 μg of Antigen A along with 10μg of CpG and 25 μg of Adju-Phos (Brenntag) as adjuvants on days 3, 6,11, 13, 17, 20 for a total of 6 boosts. The mice were bled on days 15and 22 after the 4^(th) and 6^(th) boost, respectively. Their antiserumwas assayed for antibody titers to Antigen A.

Antibody serum titers against immunogen were determined by a standardELISA. To perform the ELISA, 96-well microtiter plates (ThermoScientific) were coated at 2 μg/ml with Antigen A in phosphate-bufferedsaline (PBS, Irvine Scientific) overnight at 4° C. The next day, plateswere washed with phosphate-buffered saline containing 0.05% Tween 20(PBS-T, Sigma-Aldrich) four times using a plate washer (MolecularDevices). Plates were then blocked with 250 μl of 0.5% bovine serumalbumin (BSA, Sigma-Aldrich) in PBS and incubated for 1 hour at roomtemperature. The plates were then washed four times with PBS-T. Serafrom immunized mice and pre-immune sera were serially diluted three-foldin 0.5% BSA-PBS starting at 1:300 or 1:1000, added to the blocked platesin duplicate, and then incubated for 1 hour at room temperature. Thelast two wells were left blank to be used as a secondary antibodycontrol (background control). The plates were again washed four timeswith PBS-T in a plate washer. Goat anti-mouse IgG-Fc- Horse RadishPeroxidase (HRP) conjugated secondary antibody (Jackson Immunoresearch)was then added to the plates at 1:5000/1:10,000 dilution and incubatedfor 1 hour at room temperature. Plates were then washed eight times withPBS-T and developed using TMB/H₂O₂ as substrate. The substrate wasincubated for 20 min and the reaction was stopped with 2 N sulfuric acid(H₂SO₄, VWR, cat# BDH3500-1) or 1 N phosphoric acid (JT Baker,Cat#7664-38-2). Plates were read on a spectrophotometer (Victor, PerkinElmer) at 450 nm. Antibody titers were computed using Graphpad PRISMsoftware.

The immune response induced in mice to the injected immunogen isrepresented as antibody titers, which is defined as the reciprocal ofthe highest serum dilution at which antigen binding absorbance istwo-fold higher over background. Therefore, the higher the number, thegreater the humoral immune response to the immunogen. Antibody titersinduced to the immunogen were very high in both strains of HULC mice andin the WT mice, with no significant differences observed among thestrains (FIG. 9).

Example 3.3 Generation of pH-Sensitive Monoclonal Antibodies

When a desired immune response to the immunogen was achieved in bothstrains of HULC mice and in the WT mice, splenocytes from each mousestrain were harvested and fused with mouse myeloma cells to generatehybridoma cells, which were allowed to grow in 96-well plates. After 10days of growth, supernatants from each hybridoma cell-containing wellwere screened via immunogen-specific ELISA to identify positive antigenbinding samples. For the ELISA, 96 well micro-titer plates were coatedwith 1 μg/mL of an anti-myc polyclonal antibody (Novus Biologicals,#NB600-34) overnight at 4° C. to immobilize the myc-tagged antigen,followed by blocking with a solution of 0.5% (w/v) BSA in PBS. Theplates were washed, the antigen solutions were added to the plates at aconcentration of 1 μg/mL and allowed to bind to the coated plate for 1hour at room temperature. Subsequently, supernatants from hybridomacells were added to the wells at 1:50 dilution and allowed to bind for 1hour at room temperature. The plate bound antibodies were detected usingan anti-mouse IgG polyclonal antibody conjugated with HRP (JacksonImmunoresearch, #115-035-164). TMB substrates were added to the plates(BD Biosciences, #51-2606KC/51-2607KC) and colorimetric signals weredeveloped according to manufacturer recommended protocol. The absorbancewas recorded at 450 nm on a Victor Wallac plate reader. Antigen positivesamples defined as having an OD equal to or greater than 0.5 (with thebaseline having OD of about 0.1) were subject to affinity screeningusing a real-time surface plasmon resonance biosensor (Biacore 4000).

Kinetic binding parameters (e.g., k_(a), k_(d), K_(D), t_(1/2), etc.)for antibody binding to the immunogen at neutral pH (pH 7.4) and atacidic pH (pH 6.0) were recorded. A Biacore CM4 sensor chip wasderivatized with a polyclonal goat anti-mouse Fc antibody to captureantibodies from the supernatant. A single concentration (100 nM) ofimmunogen was then injected over the antibody-captured surface at a flowrate of 30 μl/min. Antibody-antigen association was monitored for 1.5minutes and then the dissociation of antigen from the captured antibodywas monitored for 2.5 minutes. Kinetic association (k_(a)) anddissociation (k_(d)) rate constants were determined by processing andfitting the data to a 1:1 binding with a mass transport model usingBiacore 4000 Evaluation software version 1.0. Equilibrium dissociationconstants (K_(D)) and dissociative half-lives (t_(1/2)) were calculatedfrom the kinetic rate constants as: K_(D) (M)=k_(d) k_(a); andt_(1/2)(min)=ln2/(60*k_(d)). A set of samples that displayed decreasedbinding at pH 6.0 as compared to that at pH 7.4 (pH sensitive) as wellas a set of control samples that displayed no significant rate changesbetween the pH 7.4 and pH 6.0 (pH insensitive controls) were selected tobe produced clonally. FIG. 10 depicts comparison of the number of totalantigen positives and the number of antigen positives displayingpH-sensitive antigen binding from HULC and WT mice.

Among the antigen positives, 18 and 7 clones isolated from twoheterozygous HULC1927 mice and two HULC1930 respectively, and 1 clonefrom the WT mouse, were made monoclonal. Supernatants of the monoclonalhybridomas were subject to neutral and low pH antigen dissociation rate(off-rate) analysis and cell pellets were used for light chain variabledomain DNA sequencing.

Example 3.4 Sequencing and Somatic Hypermutations in CDR3 Region ofVκ1-39Jκ5-Based Histidine Universal Light Chain Mice

Cell pellets from monoclonal hybridomas from HULC and WT mice were usedfor light chain variable domain DNA sequencing. From the 26 clones mademonoclonal (see Example 3.3 above) and subjected to sequencing, 15 wereconfirmed as using either a HULC or WT mouse light chain (MM and NN, seeTable 4). 14 clones were derived from HULC heterozygous mice (1927 or1930 mice) and 1 was derived from a WT mouse (OO, see Table 4)

From the 14 antigen positive samples derived from HULC heterozygousmice, 12 of the monoclonal antibodies utilized their corresponding HULClight chain, while 2 utilized a WT mouse light chain. All but one of theHULC utilizing antibodies retained all of the introduced histidinemutations as shown in Table 3 (italicized antibody). Sequencing of cloneAA produced 2 different HULC sequences, which is reflected by twoentries in Table 3.

TABLE 3 Number of conserved histidine insertions and somatichypermutations in light chain sequences from clones utilizing the HULClight chain Light Chain Sequences from mice utilizing HULC # Conserved #Somatic # Somatic His Hyper- Hyper- Mouse Mutations mutations inmutations Clone Name Strain in CDR3 Framework in CDRs AA 1927 4 3 0(Sequence 1) AA 1927 4 1 1 (Sequence 2) BB 1927 4 3 3 CC 1927 4 0 0 DD1927 3 1 1 EE 1927 4 2 2 FF 1927 4 0 1 GG 1927 4 1 1 HH 1927 4 2 0 II1930 3 1 1 JJ 1930 3 4 5 KK 1930 3 1 2 LL 1930 3 1 0

Example 3.5 pH-Dependent Binding of Monoclonal Antibodies Generated inVκ1-39Jκ5-Based Histidine Universal Light Chain Mice

In order to further assess the pH-dependent binding characteristics ofthe monoclonal antibodies isolated from HULC and WT mice, bindingexperiments were carried out in which the antibody/antigen associationphase was observed at neutral pH and the antibody/antigen dissociationphase was observed at either neutral or acidic pHs.

A Biacore CM4 sensor chip was derivatized with a polyclonal rabbitanti-mouse Fc antibody. Monoclonal antibody supernatants were capturedonto the anti-mouse Fc sensor surface. Two concentrations, 50 nM (induplicate) and 16.7 nM, of the immunogen were injected over themonoclonal antibody captured surface at a flow rate of 30 μl/min.Antibody-antigen association was monitored at pH 7.4 for 4 minutes andthen the dissociation of antigen from the captured monoclonal antibodywas monitored for 15 minutes at either pH 7.4 or 6.0. Dissociation(k_(d)) rate constants were determined by processing and fitting thedata using Scrubber version 2.0 curve fitting software and are shown inTable 4. Dissociative half-lives (t_(1/2)) were calculated from thedissociation rate constants as: t_(1/2) (min)=(ln2/k_(d))/60, and areshown in Table 4. Sensorgrams depicting the association/dissociationcharacteristics of several antibodies listed in Table 4 under thevarious pH conditions are shown graphically in FIG. 11. The individuallines in each graph represent the binding responses at differentconcentrations of the respective antibodies. All experiments werecarried out at 25° C. Dissociative half-life values (t½) are noted abovethe respective sensorgrams. Response is measured in RU.

TABLE 4 Dissociation (k_(d)) rate constants and dissociative half-lives(t_(1/2)) of monoclonal HULC or WT antibodies binding to their immunogenat neutral and low pH. pH 7.4 Association/pH 7.4 pH 7.4 Association/pH6.0 Dissociation Dissociation 50 nM 50 nM pH Light neutral immunogen lowimmunogen 6.0/pH 7.4 Clone chain mAb bound t½ mab bound t½ ratio Nameused capture (RU) k_(d) (1/s) (min) capture (RU) k_(d) (1/s) (min) k_(d)t½ AA HULC 129 70 5.60E−05 206 122 73 2.18E−04 53 3.9 0.3 (1927) BB HULC350 165 6.00E−04 19 378 185 2.20E−03 5 3.7 0.3 (1927) CC HULC 611 2512.03E−04 57 545 226 6.68E−03 2 33.0 0.03 (1927) DD HULC 182 75 3.55E−0433 168 74 6.44E−04 18 1.8 0.6 (1927) HH HULC 268 92 1.36E−04 85 251 915.39E−04 21 4.0 0.3 (1927) GG HULC 353 110 2.78E−04 42 328 102 8.97E−0413 3.2 0.3 (1927) FF HULC 334 202 4.79E−05 241 364 220 6.90E−05 167 1.40.7 (1927) EE HULC 339 124 5.08E−04 23 299 120 4.66E−04 25 0.9 1.1(1927) II HULC 387 174 1.22E−04 95 334 147 2.14E−04 54 1.8 0.6 (1930) JJHULC 363 14 9.83E−04 12 333 12 5.30E−04 22 0.5 1.9 (1930) KK HULC 490303 7.41E−05 156 484 295 1.29E−04 90 1.7 0.6 (1930) LL HULC 636 413.09E−04 37 597 36 5.77E−04 20 1.9 0.5 (1930) MM* WT 245 6 NA NA 203 6NA NA NA NA (from 1927 mouse) NN WT 394 231 5.26E−04 22 378 231 9.35E−0412 1.8 0.6 (from 1927 mouse) OO WT 413 89 2.94E−04 39 400 83 3.57E−04 321.2 0.8 *k_(d) and t_(1/2) values could not be determined due to lowantigen binding signal

Example 4 Engineering of Genetically Modified Mouse Comprising aHistidine-Substituted Vκ3-20Jκ1 Universal Light Chain

A mouse comprising a common Vκ3-20Jκ1 light chain was generated asdescribed in, e.g., U.S. patent application Ser. Nos. 13/022,759,13/093,156, 13/412,936, and 13/488,628 (Publication Nos. 2011/0195454,2012/0021409, 2012/0192300, and 2013/0045492, respectively), and inExample 1 above. The amino acid sequence of the germline universalVκ3-20Jκ1 light chain variable domain is set forth in SEQ ID NO:59.

Histidine substitutions were introduced into the Vκ3-20Jκ1 universallight chain targeting vector and mice generated from the same using asimilar strategy to the one described above in Example 3 for Vκ1-39Jκ5histidine modified universal light chain mice (HULC 1927 and 1930).

Briefly, the strategy for generating a histidine-modified Vκ3-20Jκ1universal light chain targeting vector is summarized in FIGS. 14A-14D. Aplasmid used for generating a targeting vector for common (universal)light chain mouse (“ULC mouse,” described in, e.g., US2011/0195454A1),containing pBS+FRT-Ub-Hyg-FRT+mouse Vκ3-7 leader+human Vκ3-20Jκ1 wasmodified by site directed mutagenesis (QuickChange Lightning Kit) toreplace Q105, Q106, Y107 and S109 or Q105, Q106 and S109 (see alignmentin FIG. 12) with histidine residues in the CDR3 region usingsite-directed mutagenesis primers shown in FIG. 13 (See FIG. 14A forthis engineering step). Resultant vectors (H105/106/107/109 andH105/106/109) were modified further and ligated into a vector comprisingmouse Igκ constant region, mouse enhancers, a mouse 3′ homology arm anda SPEC cassette (FIG. 14B). Further modification involved ligation intoa vector carrying 5′ mouse arm and comprising Frt-UB-NEO-Frt cassette(FIG. 14B). Resultant targeting vectors were electroporated into EScells comprising deletion of the mouse Igκ variable locus (comprising κvariable and joining gene segments) (FIGS. 14C-14D).

Positive ES cell clones were confirmed by using a modification of alleleassay (Valenzuela et al.) using probes specific for the engineeredVκ3-20κJ1 light chain region inserted into the endogenous κ light chainlocus. Primers and probes used in the assay are shown in Table 5 belowand set forth in the Sequence Listing; the locations of the probes aredepicted in FIGS. 14C-14D.

TABLE 5 Primers and Probes Used for ES Cell Screening Probe Probe 5′ 3′Name Assay Sequence Primer Primer Neo GOA TGGGCACA GGTGGAGA GAACACGGACAGACAA GGCTATTC CGGCATCA TCGGCTG GGC G (SEQ ID (SEQ ID (SEQ ID NO: 38)NO: 39) NO: 40) ULC-m1 GOA CCATTATG AGGTGAGG TGACAAAT ATGCTCCA GTACAGATGCCCTAAT TGCCTCTC AAGTGTTA TATAGTGA TGTTC TGAG TCA (SEQ ID (SEQ ID(SEQ ID NO: 41) NO: 42) NO: 43) 1633h2 GOA AAAGAGCC TCCAGGCA AAGTAGCT(Vκ3- ACCCTCTC CCCTGTCT GCTGCTAA 20Jκ1- CTGCAGGG TTG CACTCTGA spe-(SEQ ID (SEQ ID CT cific) NO: 65) NO: 66) (SEQ ID NO: 67) mlgKd2 Reten-GGCCACAT GCAAACAA CTGTTCCT tion TCCATGGG AAACCACT CTAAAACT TTC GGCCGGACTCCA (SEQ ID (SEQ ID CAGTAAAT NO: 47) NO: 48) GGAAA (SEQ ID NO: 49)mlgKp15 Reten- GGGCACTG CACAGCTT AGAAGAAG tion GATACGAT GTGCAGCCCCTGTACT GTATGG TCC ACAGCATC (SEQ ID (SEQ ID CGTTTTAC NO: 50) NO: 51)AGTCA (SEQ ID NO: 52)

The NEO selection cassette introduced by the targeting constructs isdeleted by transfecting ES cells with a plasmid that expresses FLP(FIGS. 14C and 14D). Optionally, the neomycin cassette may be removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Targeted ES cells described above are used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99). VELOCIMICE® independently bearing an engineeredhuman light chain gene that contains histidine residues mutated into oneor more positions along the sequence are made from the targeted ES cellsdescribed above.

Pups are genotyped and pups heterozygous for the engineeredhistidine-modified human light chain are selected for characterizingexpression of the light chain and binding capabilities of the expressedantibodies. Primers and probes for genotyping of mice specificallycomprising a universal light chain gene with either three (H105/106/109;“6183”) or four (H105/105/108/111; “6181”) histidine modifications arelisted in Table 6 below and set forth in the Sequence Listing. Micecontaining histidine modification in their universal light chains arereferred herein as “HULC” mice (histidine universal light chain mice).

TABLE 6 Primers and Probes Used for Genotyping Probe Probe 5′ 3′ NameAssay Sequence Primer Primer hVI494- GOA CTGTCATC GCAGACTG CCGAACGT 16181 ACCATGG GAGCCTGA CCAAGG (4 His) (SEQ ID AGATTTT TGAGTG mouse-NO: 68) (SEQ ID (SEQ ID spe- NO: 69) NO: 70) cific hVI495- GOA TACTGTCAGCAGACTG CCGAACGT 1 6183 TCACTAT GAGCCTGA CCAAGGTG (3 His) GG AGATTTAGTG mouse- (SEQ ID (SEQ ID (SEQ ID specific NO: 71) NO: 72) NO: 73)

Mice are immunized with antigen of interest and tested for ability togenerate antibodies with pH-dependent binding.

Example 5 Breeding of Mice Comprising a Histidine-Substituted SingleRearranged Human Universal Light Chain Mouse (HULC)

This Example describes several other genetically modified mouse strainsthat can be bred to any one of the HULC mice described herein to createmultiple genetically modified mouse strains harboring multiplegenetically modified immunoglobulin loci.

Endogenous Igλ Knockout (KO).

To optimize the usage of the engineered light chain locus, any one ofthe HULC animals described above (e.g., comprising Vκ1-39Jκ5 orVκ3-20Jκ1 histidine-substituted universal light chain) may be bred toanother mouse containing a deletion in the endogenous λ light chainlocus. In this manner, the progeny obtained will express, as their onlylight chain, the rearranged histidine-substituted human germline lightchain region as described in Examples 3 and 4 above. Breeding isperformed by standard techniques recognized in the art and,alternatively, by a commercial breeder (e.g., The Jackson Laboratory).Mouse strains bearing an engineered histidine-substituted light chainlocus and a deletion of the endogenous λ light chain locus are screenedfor presence of the unique light chain region and absence of endogenousmouse λ light chains.

Humanized Endogenous Heavy Chain Locus.

Mice bearing an engineered human germline light chain locus (HULC mice)are bred with mice that contain a replacement of the endogenous mouseheavy chain variable gene locus with the human heavy chain variable genelocus (see U.S. Pat. No. 6,596,541; the VELOCIMMUNE® mouse, RegeneronPharmaceuticals, Inc.). The VELOCIMMUNE® mouse comprises a genomecomprising human heavy chain variable regions operably linked toendogenous mouse constant region loci such that the mouse producesantibodies comprising a human heavy chain variable domain and a mouseheavy chain constant region in response to antigenic stimulation.

Mice bearing a replacement of the endogenous mouse heavy chain variableregion locus with the human heavy chain variable region locus and ahistidine-substituted single rearranged human light chain variableregion at the endogenous κ light chain locus are obtained. Reversechimeric antibodies containing somatically mutated heavy chains (humanheavy chain variable domain and mouse C_(H)) with ahistidine-substituted single human light chain (HULC, human light chainvariable domain and mouse C_(L)) are obtained upon immunization with anantigen of interest. pH-dependent human antibodies generated in suchmice are identified using antibody isolation and screening methods knownin the art or described above. Variable light and heavy chain regionnucleotide sequences of B cells expressing the antibodies, e.g.,pH-sensitive antibodies, are identified, and fully human antibodies aremade by fusion of the variable heavy and light chain region nucleotidesequences to human C_(H) and C_(L) nucleotide sequences, respectively,in a suitable expression system.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety.

What is claimed is:
 1. A method of obtaining a nucleic acid encoding a human immunoglobulin variable domain of an antigen-binding protein, comprising isolating from a B cell of a non-human animal, or a hybridoma produced from the B cell, either or both: (i) a first nucleic acid comprising a first sequence that encodes a human immunoglobulin heavy chain variable domain, and/or (ii) a second nucleic acid comprising a second sequence that encodes a human immunoglobulin light chain variable domain; wherein the non-human animal comprises in its germline genome both: (a) an immunoglobulin heavy chain locus that comprises an unrearranged human immunoglobulin heavy chain variable region gene sequence comprising human V_(H), D_(H), and J_(H) segments, and (b) an immunoglobulin light chain locus that comprises a single rearranged human immunoglobulin light chain variable region gene sequence comprising human Vκ and Jκ segment sequences, wherein the Vκ segment sequence is derived from a human Vκ1-39 or Vκ3-20 gene segment, wherein the single rearranged human immunoglobulin light chain variable region gene sequence comprises a substitution of at least one non-histidine codon of the Vκ segment sequence with a histidine codon that is expressed at a CDR3 position (according to IMGT numbering); and wherein the second sequence is derived from the single rearranged human immunoglobulin light chain variable region gene sequence.
 2. The method of claim 1, further comprising immunizing the non-human animal with an antigen of interest, and allowing the animal to mount an immune response to the antigen of interest before isolating the first and/or second nucleic acid(s), wherein the human immunoglobulin heavy chain variable domain and/or the human immunoglobulin light chain variable domain specifically binds the antigen of interest.
 3. The method of claim 2, wherein the first and/or second nucleic acid(s) comprises at least one somatic hypermutation.
 4. The method of claim 1, wherein the first nucleic acid further comprises, in operable linkage to the first sequence, an endogenous non-human immunoglobulin heavy chain constant region gene sequence and/or wherein second nucleic acid further comprises, in operable linkage to the second sequence, an endogenous non-human immunoglobulin light chain constant region gene sequence.
 5. The method of claim 4, further comprising replacing in the first nucleic acid the endogenous non-human immunoglobulin heavy chain constant region gene sequence with a human immunoglobulin heavy chain constant region gene sequence and/or replacing in the second nucleic acid the endogenous non-human immunoglobulin light chain constant region gene sequence with a human immunoglobulin light chain constant region gene sequence.
 6. The method of claim 1, wherein the non-human animal lacks a functional unrearranged immunoglobulin K light chain variable region.
 7. The method of claim 1, wherein the CDR3 position is selected from the group consisting of 105, 106, 107, 108, 109, 111 and a combination thereof (according to IMGT numbering).
 8. The method of claim 7, wherein the substitution is of one, two, three, or four CDR3 codon(s).
 9. The method of claim 1, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 gene sequence.
 10. The method of claim 9, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ1-39/Jκ5 gene sequence, and wherein the Vκ1-39/Jκ5 gene sequence comprises a replacement of at least one non-histidine codon with a histidine codon designed to express a histidine at a position selected from the group consisting of 105, 106, 108, 111, and a combination thereof (according to IMGT numbering).
 11. The animal of claim 9, wherein the single rearranged human immunoglobulin light chain variable region is derived from a rearranged Vκ3-20/Jκ1 gene sequence, and wherein the Vκ3-20/Jκ1 gene sequence comprises a replacement of at least one non-histidine codon with a histidine codon designed to express a histidine at position selected from the group consisting of 105, 106, 107, 109, and a combination thereof (according to IMGT numbering).
 12. The method of claim 1, wherein the non-human animal is a rodent selected from a rat, a mouse and a hamster.
 13. The method of 12, wherein the rodent is a rat or a mouse.
 14. The method of claim 12, wherein the rodent is a mouse.
 15. A nucleic acid obtained by the method of claim
 1. 16. A nucleic acid obtained by the method of claim
 5. 17. A cell comprising the nucleic acid of claim
 15. 18. A cell comprising the nucleic acid of claim
 16. 19. A method of obtaining a cell that expresses a human immunoglobulin variable domain with at least one histidine comprising isolating a B cell from a non-human animal that comprises in its germline genome both: (a) an immunoglobulin heavy chain locus that comprises an unrearranged human immunoglobulin heavy chain variable region gene sequence comprising human V_(H), D_(H), and J_(H) segments, and (b) an immunoglobulin light chain locus that comprises a single rearranged human immunoglobulin light chain variable region gene sequence comprising human Vκ and Jκ segment sequences, wherein the Vκ segment sequence is derived from a human Vκ1-39 or Vκ3-20 gene segment, and wherein the single rearranged human immunoglobulin light chain variable region gene sequence comprises a substitution of at least one non-histidine codon of the Vκ segment sequence with a histidine codon that is expressed at a CDR3 position (according to IMGT numbering).
 20. The method of claim 19, further comprising producing a hybridoma from the isolated B cell.
 21. A B cell obtained according to the method of claim
 19. 22. An in vitro method of making a human immunoglobulin heavy chain variable domain comprising: expressing in a host cell the first and/or second nucleic acids obtained according to the method of claim
 1. 23. An in vitro method of making a human immunoglobulin heavy chain variable domain comprising: expressing in a host cell the first and/or second nucleic acids obtained according to the method of claim
 5. 24. A human immunoglobulin heavy chain variable domain made according to the method of claim
 22. 25. A human immunoglobulin heavy chain variable domain made according to the method of claim
 23. 